Method and apparatus for detecting estradiol and metabolites thereof using an acoustic device

ABSTRACT

Methods for detecting estradiol and metabolites thereof in a sample are provided. A plurality of particles, each of which is coated with a capture agent having an affinity for estradiol, is combined with the sample to form a plurality of estradiol-particle complexes. The system also includes a transport arrangement for transporting the sample and/or particles to the sensor surface, and optionally a magnetic field inducing structure constructed and arranged to establish a magnetic field at and adjacent to the sensor surface. The resonant sensor produces a signal corresponding to an amount of estradiol-particle complexes that are bound to the sensor surface.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 11/183,484filed on Jul. 18, 2005, which claims the benefit of U.S. ProvisionalPatent Application Ser. No. 60/690,592, filed Jun. 15, 2005. Thisapplication also claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/676,759, filed on May 2, 2005, entitled “Methodsand Apparatus for Viral Load Detection and Measurement.”

TECHNICAL FIELD

The present invention relates to methods for detecting one or moreanalytes such as estradiol and metabolites thereof in fluid samples.

BACKGROUND OF THE INVENTION

Significant challenges for a system that detects analytes (e.g.,chemical and biological agents) in liquid media include concentration ofthe analyte in the media, and transport of the analyte to a sensorsurface. For biological applications, concentration issues generallyarise since the concentrations of such analytes tend to be low.Additionally, biological analytes (e.g., cells, cell fragments andmacromolecules such as proteins and nucleic acids) tend to be relativelylarge; hence, transport issues arise because these larger analytesdiffuse in fluid solution very slowly.

In addition to cells, cell fragments, and molecules such as proteins andnucleic acids, the detection of small molecule analytes can be a usefulmarker for diagnosing disease, monitoring drug pharmacokinetics in apatient, and for screening small molecule libraries for potential drugtargets. Many therapeutic drugs, including small molecule drugs, requirefrequent monitoring in patients in order to maximize the beneficialeffects of the drug and avoid adverse effects that may result.

For example, estradiol is a small molecule that is used to assess anumber of health conditions. For example, blood tests for folliclestimulating hormone (FSH) and estradiol (E2) can be used to checkovarian reserves and for adequate egg function. In addition, estradioland metabolites thereof can be used as a risk factor determinant forbreast cancer. An elevated level of estradiol is correlated withincreased risk of developing breast cancer. However, a low estradiollevel is correlated with an increased risk for vertebral fractures. TheU.S. Food and Drug Administration has approved the use of the drugtamoxifen in the prevention of breast cancer in certain patients.However, tamoxifen can lower serum estradiol levels to a point thatincreases the incidence of vertebral fracture. It is therefore desirablefor a physician to be able to frequently monitor estradiol levels in apatient to ensure that estradiol levels remain within a certain range toreduce the risk of breast cancer while not increasing the risk ofvertebral fracture.

Therapeutic drugs also require frequent monitoring in patients.Therapeutic drugs include, for example, immunosuppressant medicationssuch as cyclosporin, tacrolimus (FK-506), rapamycin, and mycophenolicmofetil. Immunosuppressants have a narrow therapeutic index and highdegree of inter- and intra-patient variability in bioavailability.Immunosuppressants require careful, frequent monitoring in patientsamples in order to balance the level of immunosuppression needed toprevent transplant rejection while avoiding the adverse effects ofexcessive immunosuppression such as toxicity, infection, and increasedrisk of developing cancer.

The detection of small molecules such as estradiol is often difficultnot only because of potentially low levels in the sample, but alsobecause of the limited number of suitable capture agents that can beused to bind the small molecule analyte. As a result of suchlimitations, it is often difficult to design or modify existing assays,such as enzyme-linked immunosorbant assays that are sensitive andspecific enough to detect low levels of the small molecule analyte.

Typically, detection of analytes in patient samples requires obtainingthe sample in the doctor's office or clinic and sending the sample offsite for analysis. Depending on the analyte, the analysis can take oneday to several weeks. The results of the analysis are transmitted to thedoctor, who then uses the information to adjust treatment as necessary,and contacts the patient to convey the new treatment regimen. The delayassociated with analyzing a sample makes it difficult for a doctor toaccurately specify a proper treatment.

There is a need for improved assays that can be used to more readilydetect small molecule analytes, and to detect low concentrations ofanalyte. In addition, there is a need for improved measurement ofanalytes including small molecule analytes in order to customize drugregimens to maintain efficacy of the drug while reducing unwanted sideeffects in individual patients. Furthermore, there is a need for methodsand apparatus that can be used at the point of care to measurebiologically and/or clinically relevant analytes in order to reduce thedelay between obtaining the sample and obtaining the results of theassay.

A key metric for competitive detection is the amount of analyteaccumulated on a sensor per unit time. For good performance, the rate ofaccumulation (and the resulting signal transient) needs to be fastrelative to the sensor drift rate. Another key performance metric for ananalyte detection system is the degree to which the system canpreferentially collect the analyte of interest on the sensor surface.Since many biological samples contain extraneous background components(e.g., other proteins, cells, nucleic acids, dirt), it is necessary toprevent these background components from interfering with the desiredmeasurement. So, a transport method that selectively draws the analyteto the sensor and allows interfering background components to pass byhas definite advantages. Such a method used in concert with selectivebinding of the analyte (e.g., antibody, complimentary DNA strands, etc.)to the sensor surface can deliver high sensitivity measurements forsamples with large amounts of extraneous background components relativeto the amount of analyte.

Various methods for improving transport of analyte to a sensor surfacehave been proposed, including filtration, novel flow geometries,acoustic fields, electrical fields (time varying and static) andmagnetic fields.

Acoustic excitation has been used to draw cells to field nodes, but itis difficult to use this technique alone to transport material to asurface.

Electrical fields (electrophoresis and dielectrophoresis) have been usedto enhance transport but are not universally applicable to all analytesand sample types. They are generally more effective for larger analytes(e.g., cells). Furthermore, the electrical properties of microbes canvary within a given species and strain, making it hard to predict systemperformance under all intended operating conditions. Sometimes it isnecessary to tailor the ionic strength of the sample to improve theperformance of the transport. This requirement can conflict with theoptimum binding or wash conditions in an assay. Also, electrical fieldscan dissipate energy and heat conductive fluids (e.g., 0.1 M phosphatebuffer solution), which is undesirable since heating can damage thebiological analytes.

Immunomagnetic separation (IMS) methods are known in the art forisolating analyte from a sample.

SUMMARY OF THE INVENTION

The present invention is drawn to methods for detecting whether one ormore analytes is present in a sample. In one embodiment, the methodcomprises the steps of introducing a plurality of magnetic particlescoated with a capture agent into a fluid chamber, wherein at least onesurface of the fluid chamber comprises an acoustic device having a firstanalyte bound thereto. The plurality of magnetic particles can becontacted with the sample prior to introducing the particles into thechamber, or the sample can be introduced into the fluid chamber prior toor simultaneously with the introduction of the particles. A magneticflux can be created in proximity to the acoustic device to attract atleast one of the plurality of magnetic particles toward said surface.Signal output by said acoustic device is monitored, thereby detectingwhether one or more analytes is present in the sample.

In another embodiment, the method for detecting whether one or moreanalytes is present in a sample comprises the steps of introducing aplurality of particles and a competitor molecule into a fluid chamber,said particles being coated with a first capture agent capable ofbinding the analyte. In certain embodiments, the particles can bemagnetic particles. At least one surface of the fluid chamber comprisesan acoustic device that has been coated with a second capture agentcapable of binding to the competitor molecule. A magnetic flux can becreated in proximity to the acoustic device to attract at least one ofthe plurality of magnetic particles toward said surface, and signaloutput by said acoustic device is monitored, thereby detecting whetherone or more analytes is present in the sample. The plurality ofparticles can be contacted with the sample and competitor molecule priorto introducing the particles into the chamber, or the sample andcompetitor molecule can be introduced into the fluid chamber prior to orsimultaneously with the introduction of the particles. Signal output bysaid acoustic device is monitored, thereby detecting the analyte.

The present invention is also drawn to methods for determining whetherto adjust drug dosage in an individual. The method comprises detecting alevel of one or more analytes in a sample. In one embodiment, the methodof detecting a level of one more analytes in a sample comprisesintroducing a plurality of magnetic particles coated with a captureagent into a fluid chamber, wherein at least one surface of the fluidchamber comprises an acoustic device having a first analyte boundthereto as described above. A magnetic flux is created in proximity tothe acoustic device to attract at least one of the plurality of magneticparticles toward said surface. Signal output by said acoustic device ismonitored, thereby detecting the level of one or more analytes in thesample. Whether to adjust the dosage of the drug is determined based onthe level of the one or more analytes in the sample.

In still another embodiment, the method for detecting an analyte capableof binding a capture agent comprises introducing a plurality ofparticles into a fluid chamber. Each magnetic particle is coated with adifferent analyte and at least one surface of the fluid chambercomprises an acoustic device having a capture agent bound thereto.Signal output by said acoustic device is monitored, thereby detectingthe analyte capable of binding a capture agent The plurality of magneticparticles can be contacted with the sample prior to introducing theparticles into the chamber, or the sample can be introduced into thefluid chamber prior to or simultaneously with the introduction of theparticles. Signal output by said acoustic device is monitored, therebydetecting the analyte.

The present invention is drawn to methods for detecting estradiol in asample. In one embodiment, the method comprises the steps of introducinga plurality of particles coated with a capture agent capable of bindingestradiol into a fluid chamber, wherein at least one surface of thefluid chamber comprises an acoustic device having estradiol boundthereto. The plurality of magnetic particles can be contacted with thesample prior to introducing the particles into the chamber, or thesample can be introduced into the fluid chamber prior to orsimultaneously with the introduction of the particles. Signal output bysaid acoustic device is monitored, thereby detecting estradiol in thesample.

In another embodiment, the method comprises the steps of introducing aplurality of particles and a competitor molecule into a fluid chamber,said particles being coated with a first capture agent capable ofbinding estradiol. At least one surface of the fluid chamber comprisesan acoustic device that has been coated with a second capture agentcapable of binding to the competitor molecule. The plurality of magneticparticles can be contacted with the sample prior to introducing theparticles into the chamber, or the sample can be introduced into thefluid chamber prior to or simultaneously with the introduction of theparticles. Signal output by said acoustic device is monitored, therebydetecting estradiol.

The present invention is drawn to methods for detecting one or moreimmunosuppressants in a sample. The method comprises the steps ofintroducing a plurality of particles coated with a capture agent capableof binding the immunosuppressant into a fluid chamber, wherein at leastone surface of the fluid chamber comprises an acoustic device havingsaid immunosuppressant bound thereto. The plurality of particles can becontacted with the sample prior to introducing the particles into thechamber, or the sample can be introduced into the fluid chamber prior toor simultaneously with the introduction of the particles. Signal outputby said acoustic device is monitored, thereby detecting theimmunosuppressant in the sample.

In another embodiment, the method for detecting one or moreimmunosuppressants comprises introducing a plurality of particles and acompetitor molecule into a fluid chamber, said particles being coatedwith a first capture agent capable of binding an immunosuppressant,wherein at least one surface of the fluid chamber comprises an acousticdevice that has been coated with a second capture agent capable ofbinding to the competitor molecule. The plurality of particles can becontacted with the sample prior to introducing the particles into thechamber, or the sample can be introduced into the fluid chamber prior toor simultaneously with the introduction of the particles. Signal outputby said acoustic device is monitored, thereby detecting theimmunosuppressant.

The present invention provides improved measurement of analytes. Incertain embodiments, the analytes can include small molecules includingbut not limited to estradiol and therapeutic drugs. As a result of thepresent invention, the pharmacokinetics of a given drug can be readilydetermined and drug regimens can be more readily customized (by, forexample, a physician) to maintain efficacy of the drug while reducingunwanted side effects. In addition, the present invention can be used atthe point of care to measure biologically and/or clinically relevantanalytes while avoiding delays (associated with, for example, sendingsamples to an off-site testing facility) in order to customize care andincrease the level of patient compliance. As used herein, the term pointof care can include, for example, the doctor's office, clinic, emergencyroom, and mobile treatment facility (e.g., an ambulance). In oneembodiment, the method further comprises adjusting dosage of saidtherapeutic drug administered to an individual based on the level ofsaid drug in the sample. The level of the therapeutic drug can be variedbased on the known therapeutic range for the therapeutic drug beingtested.

The present invention can be used to diagnosis disease or assess therisk of developing a disease based on the level of analyte detected in asample. In one embodiment, the disease is breast cancer. The method ofdetecting a breast cancer marker can be used, for example in conjunctionwith other diagnostic tests such as a mammogram, to detect breast cancerin an individual. In another embodiment, the method of detecting abreast cancer marker can be used to determine the likelihood that theindividual will develop breast cancer. In addition, the invention can beused to assess a condition of the patient, for example, fertility of apatient can be determined based on the estradiol level. The presentinvention can be used to monitor the level of biologically or clinicallyrelevant substances in a patient. Furthermore, the present invention canbe used to determine whether a patient is eligible to receive a drug, oreligible to be included in a drug study, based on the level of a givenanalyte in the patient.

The efficiency and low operating cost associated with the system 100 ofFIG. 1 is well-suited for running individual samples in a manner thatdoes not require operating in a batch mode where many samples areacquired prior to running an assay. In this manner, embodiments of thepresent invention are well suited for analyzing samples on an individualbasis.

In one embodiment of an analyte detection system, an analyte binds to amagnetic particle (e.g., a magnetic bead) to form an analyte-particlecomplex. The analyte-particle complex is transported and localized ontothe surface of a sensing device by applying a gradient magnetic field.The magnetic field induces a polarization in the magnetic material ofthe particle that is aligned with the local magnetic field lines. Theparticle experiences a net force in the direction of the gradient,causing the particle to migrate toward regions of higher field strength.The magnetic field distribution is tailored to draw analyte-particlecomplexes from a sample flow and distribute them across the surface ofthe sensing device. The extraneous, background components of the sample(e.g., cells, proteins) generally have a much lower magneticsusceptibility as compared to the magnetic particles, and so themagnetic field does not significantly influence them. Hence, only a verysmall fraction of this background material interacts with the sensorsurface.

In one embodiment, the sensing device is a flexural plate wave (FPW)device, which functions particularly well with the magnetic particlesfor two reasons. First, the presence of the magnetic particles on thesurface of the sensing device results in an amplified FPW signalresponse. The larger combined size and density of the analyte-particlecomplex yields a larger FPW signal response than the analyte alone.Second, the surface of the sensor in the FPW device consists of a thinmembrane that is typically only a few micrometers thick, which allowslarger magnetic fields and field gradients to be created at the sensorsurface because the field source can be positioned closer to the sampleflow. This results in higher fractional capture of the analyte from thesample. With this higher capture rate and efficiency, it is possible toprocess larger sample volumes in shorter times than would be otherwisepossible.

In one aspect, an apparatus for detection of an analyte includes a fluidchamber having at least one opening for fluid to enter, and a flexuralplate wave device defining at least a portion of at least one interiorsurface of the fluid chamber. The apparatus further includes amonitoring device to monitor at least one signal output by the flexuralplate wave device, a plurality of magnetic particles coated with acapture agent having an affinity for the analyte, and a first source ofmagnetic flux to selectively attract magnetic particles to the at leastone interior surface of the fluid chamber.

In another aspect, a cartridge for a resonant device system includes afirst fluid chamber having at least one opening for fluid to enter, anda flexural plate wave device defining at least one interior surface ofthe fluid chamber. The apparatus further includes a first source ofmagnetic flux to selectively attract magnetic particles to the at leastone interior surface of the first fluid chamber.

In another aspect, a method for detection of an analyte includescombining a fluid containing an analyte with a plurality of magneticparticles that comprise a capture agent having an affinity for theanalyte to produce at least some magnetic particles bound to at leastsome analyte. The method further includes directing the combined fluidinto a first fluid chamber, wherein at least one surface of a flexuralplate wave device is in fluid communication with the fluid in the firstfluid chamber. The method also includes creating a first magnetic fluxin proximity to the flexural plate wave device to magnetically attractat least some of the bound magnetic particles to the at least onesurface of the flexural plate wave device.

In another aspect, a method for detection of an analyte includes coatingat least a portion of a surface of a flexural plate wave device locatedin a fluid chamber with a first capture agent, and directing a fluidcontaining an analyte into the fluid chamber to bind some of the analyteto the capture agent located on the flexural plate wave device. Themethod further includes directing a fluid containing a plurality ofmagnetic particles that comprise a second capture agent into the fluidchamber, and creating a magnetic flux in proximity to the flexural platewave device to attract at least some of the magnetic particles towardsthe surface of the flexural plate wave device.

As a result of the present invention, much lower levels of analyte canbe detected compared to conventional assays. The detection limit ofanalyte is improved by a number of factors. In one embodiment, thepresent invention uses fewer particles per sample, thereby resulting inparticles that are more densely coated with analyte, even where theconcentration of analyte in the sample is low. As a result of thepresence of higher levels of analyte per particle, each coated particlehas a higher avidity for the sensing surface.

Where the assay format is a competition assay, that is, the sensingsurface is coated with the analyte, particles that are more denselycoated with analyte are more effectively blocked from binding to thesensing surface because each particle comprises fewer capture agentmolecules that are free to bind to the analyte on the sensing surface.In other words, a lower particle to analyte ratio results in bettercompetition between the analyte in the sample and the analyte on thesensing surface which allows for the detection of lower concentrationsof analyte in the sample. Thus, the present invention improves theaccuracy and the sensitivity of the system because the particles aremore densely coated with analyte, and have a higher binding strength forthe sensing surface.

In another embodiment, devices fabricated and methods performedaccording to the principles of the present invention are particularlyuseful for capturing low concentrations of particles because the sensorsurface is thin (for example, in one embodiment, on the order of severalmicrons). In one embodiment, the thin surface is used in conjunctionwith a magnetic field gradient and magnetic particles. Because thesensor surface is thin, a large magnetic field gradient can be inducedone the side of the sensor surface. The large magnetic field gradientcan enhance the attraction of the particles to the sensor surface byfocusing the field close to the surface of the sensor, while allowingother material contained in the sample to flow by. The large magneticfield gradient can enhance the attraction of the magnetic particles tothe sensor surface by focusing the field close to the surface of thesensor, while allowing other material contained in the sample to flowby. In this way, lower numbers of particles can be used, therebyimproving the detection limit of the system, because the magnetic fieldgradient serves to concentrate the particles near the sensing surfacewhere they can interact with the bound analyte or capture agent,depending on the assay format. In various embodiments, the fieldgradient can be removed to allow any non-specifically bound particles tobe washed away. In various embodiments, the magnetic field gradient canbe removed to allow any non-specifically bound particles to be washedaway.

BRIEF DESCRIPTION OF DRAWINGS

The foregoing and other objects of this invention, the various featuresthereof, as well as the invention itself, may be more fully understoodfrom the following description, when read together with the accompanyingdrawings in which:

FIG. 1A shows one embodiment of an analyte detection system, constructedaccording to the invention.

FIG. 1B shows another embodiment of a portion of the analyte detectionsystem shown in FIG. 1A.

FIG. 2 shows a more detailed view of the FPW sensor shown in FIG. 1A.

FIG. 3 shows a general detection protocol for using an FPW sensor incombination with analyte-particle complexes for detection of biologicalanalytes.

FIG. 4 shows the change in the signal from multiple FPW sensors as afunction of time for an exemplary detection protocol.

FIG. 5 is a summary plot showing the final signal change detected as afunction of original analyte concentration.

FIG. 6 shows a time evolution plot for a detection protocol, similar tothe plot shown in FIG. 4, but rather for a PSA analyte.

FIG. 7 is a schematic of three different formats for attaching ananalyte to the sensing surface of the acoustic device.

FIG. 8 is a schematic of one embodiment of the present invention, wherecompetitor molecule (e.g., analyte) is bound to the sensing surface.

FIG. 9 is a schematic of one embodiment of the present invention, wherethe competitor molecule is the analyte of interest linked to a tag andthe sensing surface of the acoustic device is coated with a captureagent that is capable of binding to the tag.

FIG. 10 is a schematic of one embodiment of the present invention, wherethe competitor molecule comprises a carrier and two or more analytemolecules of interest bound thereto, and the sensing surface of theacoustic device is coated with a capture agent that is capable ofbinding to the analyte of interest.

FIG. 11 shows the change in signal from multiple FPW sensors as afunction of PSA concentration.

FIG. 12 shows the change in signal from multiple FPW sensors as afunction of estradiol concentration in serum.

FIG. 13 is a dose response curve of FK-506 in buffer.

FIG. 14 shows the change in signal from multiple FPW sensors as afunction of FK-506 concentration in buffer.

FIG. 15 is a dose response curve of FK-506 in serum.

FIG. 16 shows the change in signal from multiple FPW sensors as afunction of FK-506 concentration in serum.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is drawn to methods of detecting analytes,including small molecule analytes, using an acoustic device. Typically,small molecule analytes contain a single or only a few binding sitesthat can be recognized by capture agents. Because a small molecule has afew or only one binding site, one embodiment of the present inventionuses competitive binding to detect and/or quantify the analyte. Wherethe analyte has binding sites that can be recognized by two or morecapture agents, the analyte can be detected, for example, using asandwich assay with two different capture agents.

Increased endogenous estradiol is clinically correlated with theprevalence of breast cancer in women. It has been shown, for example,that women with increased mammographic breast density, a clinicalmanifestation of excessive breast tissue estrogen synthesis, are at ahigher risk of breast cancer (Boyd, N. F. et al., N. Engl. J. Med.,347:886-94 (2002)). In addition, it has been shown that women withlevels of serum estradiol levels in the upper quartile are at higherrisk of developing breast cancer than postmenopausal women with normalserum estradiol levels in the lower quartile (Cauley, J. A. et al., Ann.Intern. Med., 130(4 Pt 1):270-7 (1999)). Therefore, the method ofdetecting estradiol can be used to detect breast cancer. In addition,the method of the present invention can be used in conjunction withother diagnostic tests such as a mammogram, to detect breast cancer inan individual. In another embodiment, the method of detecting estradiolcan be used to determine the likelihood that the individual will developbreast cancer.

The method of detecting estradiol can be used to assess or develop atreatment plan for the individual or to determine whether an individualis suitable or eligible for a particular therapy, treatment, or clinicalstudy. For example, the method can be used to assess whether theindividual is a candidate for treatment with tamoxifen. In anotherembodiment, estradiol levels can be used to monitor hormone replacementtherapy in a patient in order to balance the benefits of hormonereplacement with the risks of elevated estradiol levels.

In general terms, an analyte is detected based on the ability ofparticles coated with a capture agent (also referred to herein as afirst capture agent) to alter the resonating frequency of an acousticdevice. The capture agent is capable of binding to the analyte. In oneembodiment, a plurality of particles coated with a capture agent isexposed to the sensing surface of the acoustic device in the presence ofthe sample and a competitor molecule. The sensing surface can be part ofa fluid chamber or channel as described herein. In addition, the coatedparticles can be exposed to the sensing surface in a static mode, forexample, the coated particles can be introduced into the chamber andincubated for a given period of time. In another embodiment, the coatedparticles can be exposed to a sensing surface in a non-static mode, forexample, by flowing the coated particles through the fluid chamber orchannel.

As described herein, it was surprisingly found that using a lowerconcentration of particles allowed for the detection of lowerconcentrations of analytes. The finding was unexpected becauseconventional wisdom, in assays involving capture by particles, calls forthe use of a high quantity of particles in order to maximize captureefficiency.

Generally, in the competitive binding format of the present invention,the amount of particles capable of binding to the sensing surface of theacoustic device is inversely proportional to the amount of analyte ofinterest present in the sample. Higher levels of analyte in the sampleresults in more of the capture agent on the particles being occupied byanalyte from the sample, and fewer capture agents on the particles beingavailable to bind to the competitor molecule, thereby affecting thenumber or amount of particles that can interact with the sensing surfaceof the acoustic device. As described below, the competitor molecule canbe present in solution or can be bound to the sensing surface of theacoustic device. In a preferred embodiment, the particles are magnetic,and a magnetic flux is applied in proximity to the acoustic device toattract at least one of the plurality of magnetic particles toward thesensing surface.

In one embodiment, a method for detecting one or more analytes comprisesintroducing a plurality of particles coated with a capture agent capableof binding the analyte into a fluid chamber, wherein at least onesurface of the fluid chamber comprises an acoustic device having acapture agent capable of binding the analyte bound thereto. This formatis referred to herein as a sandwich format. The plurality of particlescan be contacted with the sample prior to introducing the particles intothe chamber or the sample can be introduced into the chamber prior to orsimultaneously with the introduction of the particles. Signal output bysaid acoustic device is monitored, thereby detecting one or moreanalytes in the sample.

Assay Formats

While not wishing to be bound by theory, in the case of a small moleculeanalyte, where the assay format is a competition assay, that is, thesensing surface is coated with the analyte, use of few particles or alower concentration of particles allows the particles to become moredensely coated with analyte. Particles that are more densely coated withanalyte are more effectively blocked from binding to the sensing surfacebecause each particle comprises fewer capture agent molecules that arefree to bind to the analyte on the sensing surface. In other words, alower particle to analyte ratio results in better competition betweenthe analyte in the sample and the analyte on the sensing surface whichallows the detection of lower concentrations of analyte in the sample.The particles can be used in a concentration from about 1×10² to about1×10⁷ per mL. In another embodiment, the particles are at aconcentration of about 5×10³ to about 5×10⁵ per mL. FIG. 7 shows threeembodiments where the sensing surface of the acoustic device is coatedwith an analyte. In panel, B, the analyte 10 is bound directly to thesurface 12 via a chemical linker. In panels A and C, the analyte isindirectly bound to the surface. In panel A, the surface, 12 is coatedwith a first member of a binding pair 32. The second member of thebinding pair 22, having one or more analyte molecules 10 bound theretois allowed to bind to the first member of the binding pair coating thesurface. In panel C, the surface, 12 is coated with a first member of abinding pair. The second member of the binding pair 22, is allowed tobind to the surface bound first member of the binding pair, and captureagent 16 labeled with the first member of a binding pair 32 is allowedto bind the second member of the binding pair. Competitor molecule 24comprising a carrier having one or more analyte molecules bound theretois allowed to bind to the capture agent 16.

As shown in FIG. 8, in one embodiment, the competitor molecule comprisesthe analyte of interest 10 bound to the sensing surface 12 of theacoustic device (panel A). Particles 14 coated with capture agent 16 areexposed to sample containing the analyte 18 (panel B). Particle-boundcapture agent that has not bound analyte from the sample 20 is free tobind the analyte bound to the surface of the acoustic device (panel C).As shown in panels C and D, higher levels of analyte in the sampleresult in fewer particles binding the acoustic device sensing surfacebecause more of the capture agent present on the particles is occupiedwith analyte from the sample. Signal output by the acoustic device ismonitored to determine the amount and/or number of particles that havebound to the surface, thereby detecting whether one or more analytes ispresent in a sample. In addition, the presence or amount of analytepresent in the sample can be determined, for example by comparing thesignal to a control. In an alternative embodiment, as described herein,the particles can be magnetic particles. After introducing the particlesinto the fluid chamber, magnetic flux is created in proximity to theacoustic device to attract at least one of the plurality of magneticparticles toward the sensing surface (similarly as described, forexample, in FIGS. 1A, 1B and 2).

In another embodiment, as shown in FIG. 9, the sensing surface 12 of theacoustic device is coated with a capture agent 22 (also referred toherein as a second capture agent) that is capable of binding to thecompetitor molecule 24. The competitor molecule can be, for example, theanalyte of interest 10 bound to a tag 26, and the second capture agentis capable of binding to the tag. As shown in FIG. 9, higher levels ofanalyte in the sample results in fewer particles 14 binding to thesensing surface of the acoustic device because more of the capture agentpresent on the particles 16 is occupied with analyte from the sample.Because the second capture agent is capable of binding to the tagportion of the competitor molecule, the coated particles bind to thesensing surface of the acoustic device only if the particles have boundthe competitor molecule.

In another embodiment, as shown in FIG. 10, the competitor molecule 24can be, for example, two or more analyte molecules or moieties ofinterest 10 bound to a carrier 28 and the second capture agent 40 iscapable of binding the analyte. As shown in FIG. 10, the second captureagent can be indirectly bound to the acoustic device sensing surface.The surface-bound and particle-bound capture agents can be the samecapture agent. Particles 14 coated with capture agent 16 are exposed tosample containing the analyte 10 and the competitor molecule 24 (panelB). Particle-bound capture agent that that has not bound analyte fromthe sample is free to bind the competitor molecule, which in turn isbound by the capture agent that is bound to the sensing surface (panelC). As shown in panel D, higher levels of analyte present in the sampleresults in fewer particles binding the sensing surface of the acousticdevice because more of the capture agent present on the particles isoccupied with analyte from the sample. Where the analyte is a smallmolecule having only one copy of a given epitope or binding site, thecoated particles will bind to the acoustic device sensing surface onlyif the particles have bound to the competitor molecule.

The present invention can be used to screen for one or more smallmolecules of interest. In this embodiment, a plurality of particles isintroduced into a fluid chamber. The plurality of particles includesparticles coated with different analytes. At least one surface of thefluid chamber comprises an acoustic device having a capture agent boundthereto, wherein the capture agent is capable of binding the analyte ofinterest. Signal output by said acoustic device is monitored, therebydetecting an analyte capable of binding the capture agent.

The method of the present invention can be used to detect the presenceof the analyte and/or to determine the concentration or level of theanalyte in the sample. The concentration detected or measured by themethod of the present invention can be an absolute concentration or arelative concentration. For example, the concentration may be a ratiorelative to the concentration of a reference analyte present in the samesample of body fluid. The concentration can be obtained by comparing thedata with similar data obtained at a different time, to determinewhether a significant change in actual concentration has occurred. Inany of the embodiments described herein, the read-out can be provided bythe method as a positive or negative read-out. In other embodiments, themethod can provide a positive read-out if a certain threshold level ofanalyte is detected.

For each of the embodiments described herein, a number of variations canbe made to the method as described below, without departing from thescope of the invention.

Samples

Samples suitable for use in the present invention includes any materialsuspected of containing the analyte. The sample can be used directly asobtained from the source or following one or more steps to modify thesample. In one embodiment, the sample is a biological sample. The samplecan be derived from any biological source, such as a physiological fluid(e.g., blood, saliva, sputum, plasma, serum, ocular lens fluid,cerebrospinal fluid, sweat, urine, milk, ascites fluid, synovial fluid,peritoneal fluid, amniotic fluid, and the like) and fecal matter. Thesample can be obtained from biological swabs such as nasal or rectalswabs. In addition, the sample can be biopsy material. The sample can beobtained from a human, primate, animal, avian or other suitable source.

The sample can be pretreated prior to use, such as preparing plasma fromblood, diluting viscous fluids, and the like. The sample can also befiltered, distilled, extracted, or concentrated. For example, some smallmolecule drugs permeate blood cells and bind to proteins in blood (e.g.,FK506). Small molecules can be extracted by adding a proteinprecipitation agent and cell lysing agent. For example, a mixture ofzinc sulphate, polyethylene glycol and methanol can be added to bloodand the solvent phase eluted after centrifugation. An alternativepreparation method is to add a mixture containing protein digestionenzymes and detergents to break down the proteins and cells to releasethe small molecule. After centrifugation, the liquid phase is eluted andanalyzed to determine the small molecule concentration in the originalsample. The sample can also be treated to inactivate or modify certainactivities in the sample capable of interfering with the analyte or thedetection process. For example, a decomplexing antagonist can be addedto the sample to disassociate the analyte from other molecules that maybe bound to and/or may interfere with the ability of the capture agentto bind to the analyte. Such antagonsists can be, for example, steroidantagonists. In the case of estradiol detection, the sample can betreated by adding danazol to disassociate estradiol from sex hormonebinding protein.

Other liquid samples besides physiological fluids can be used, such aswater, food products, and the like, for the performance of environmentalor food production assays. In addition, a solid material suspected ofcontaining the analyte can be used as the test sample. A solid testsample can be modified (e.g., homogenized, extracted, or solubilized) toform a liquid medium or to release the analyte.

The sample volume can be as little as 10 μL or as much as 250 mL. In oneembodiment, the sample volume can be as little as 50 μL or as much as 5mL. In one embodiment, the sample volume is about 1 to about 5 mL.

In one embodiment, a single sample can be run on a single cartridge asdescribed below. In addition, in order to test a panel of one or moreanalytes, a single sample can be divided into two or more aliquots. Eachaliquot can be tested for a different analyte, for example, using adifferent cartridge for each analyte to be tested. In this way, samplescan be analyzed on an individual basis, that is, as they are acquiredfrom the patient, rather than in batch mode, e.g, accumulating multiplesamples to analyze at the same time or in the same machine run.

Capture Agents

Suitable capture agents for use in the present invention include anymolecule capable of binding to an analyte of interest. The term “captureagent” includes molecules or multi-molecular complexes that can bind toan analyte of interest. Capture agents preferably bind to their bindingpartners in a substantially specific manner. Capture agents withdissociation constants (KD) of less than about 10⁻⁶ are preferred. Thecapture agent can also be, for example, polypeptides, nucleic acids,carbohydrates, nucleoproteins, glycoproteins, glycolipids andlipoproteins. Antibodies or antibody fragments are highly suitable ascapture agents. Antibodies capable of binding to the analyte of choicemay be obtained commercially or may be prepared using standard methodsfor generating antibodies.

Antigens may also serve as capture agents, since they are capable ofbinding antibodies. A receptor which binds a ligand is another exampleof a possible capture agent. Protein-capture agents are understood notto be limited to agents which only interact with their binding partnersthrough noncovalent interactions. Capture agents may also optionallybecome covalently attached to the proteins which they bind. For example,the capture agent may be photocrosslinked to its binding partnerfollowing binding.

The term “antibody” includes any immunoglobulin, whether naturallyproduced or synthetically produced in whole, or in part. Derivatives ofantibodies that maintain the ability of the antibody to bind to theanalyte of interest are also included in the term. The term alsoincludes any protein having a binding domain which is homologous orlargely homologous to an immunoglobulin binding domain. These proteinsmay be derived from natural sources, produced synthetically in whole orin part. An antibody may be monoclonal or polyclonal. The antibody maybe a member of any immunoglobulin class, including IgG, IgM, IgA, IgD,and IgE. Where the analyte is known to bind a carrier protein, theantibody can be specific for the free form of the analyte or thecarrier-bound form of the analyte. Antibodies that are capable ofbinding an analyte of choice can be obtained commercially or producedusing known methods for generating antibodies.

The term antibody also includes antibody fragments. The term “antibodyfragments” refers to any derivative of an antibody which is less thanfull-length. Preferably, the antibody fragment retains at least theability to bind the analyte of interest. Examples of antibody fragmentsinclude, but are not limited to, Fab, Fab′, F(ab′)₂, scFv, Fv, dsFvdiabody, and Fd fragments. The antibody fragment may be produced by anymeans. For instance, the antibody fragment may be enzymatically orchemically produced by fragmentation of an intact antibody or it may berecombinantly produced from a gene encoding the partial antibodysequence. Alternatively, the antibody fragment may be syntheticallyproduced in whole or in part. The antibody fragment may optionally be asingle chain antibody fragment. Alternatively, the fragment may comprisemultiple chains which are linked together, for instance, by disulfidelinkages. The fragment may also optionally be a multimolecular complex.A functional antibody fragment will typically comprise at least about 50amino acids and more typically will comprise at least about 200 aminoacids.

Single-chain Fvs (scFvs) are recombinant antibody fragments consistingof only the variable light chain (V_(L)) and variable heavy chain(V_(H)) covalently connected to one another by a polypeptide linker.Either V_(L) or V_(H) may be the NH₂-terminal domain. The polypeptidelinker may be of variable length and composition so long as the twovariable domains are bridged without serious steric interference.Typically, the linkers are comprised primarily of stretches of glycineand serine residues with some glutamic acid or lysine residuesinterspersed for solubility. “Diabodies” are dimeric scFvs. Thecomponents of diabodies typically have shorter peptide linkers than mostscFvs and they show a preference for associating as dimers. An “Fv”fragment is an antibody fragment which consists of one V_(H) and oneV_(L) domain held together by noncovalent interactions. The term “dsFv”is used herein to refer to an Fv with an engineered intermoleculardisulfide bond to stabilize the V_(H)-V_(L) pair. A “F(ab′)₂” fragmentis an antibody fragment essentially equivalent to that obtained fromimmunoglobulins (typically IgG) by digestion with an enzyme pepsin at pH4.0-4.5. The fragment may be recombinantly produced. A “Fab”′ fragmentis an antibody fragment essentially equivalent to that obtained byreduction of the disulfide bridge or bridges joining the two heavy chainpieces in the F(ab′)₂ fragment. The Fab′ fragment may be recombinantlyproduced. A “Fab” fragment is an antibody fragment essentiallyequivalent to that obtained by digestion of immunoglobulins (typicallyIgG) with the enzyme papain. The Fab fragment may be recombinantlyproduced. The heavy chain segment of the Fab fragment is the Fd piece.

Suitable polypeptide capture agents also include virtually any peptide,polypeptide, or protein that is capable of binding to an analyte ofinterest, or a small molecule such as a small organic molecule. In oneembodiment, the capture agent is an antibody that is capable of bindingto the analyte of interest. In preferred embodiments, the capture agentis capable of binding to a small molecule. Suitable polypeptide captureagents may be obtained, for example, commercially, using recombinantmethods, using synthetic production methods, or by purification from anatural source. Polypeptides include, for example, cell surface andsoluble receptor proteins, such as lymphocyte cell surface receptors,steroid receptors, nuclear proteins, signal transduction molecules,transcription factors, allosteric enzyme inhibitors, clotting factors,enzymes (e.g., proteases and thymidylate synthetase, serine/threoninekinases, threonine kinases, phosphatases, bacterial enzymes, fungalenzymes and viral enzymes), proteins associated with DNA and/or RNAsynthesis or degradation and the like. As described in more detailbelow, where more than one capture agent is used, the capture agents canbe, for example, isoforms of each other.

The capture agent can also be a nucleic acid such as RNA or DNA, orpeptide nucleic acid. In one embodiment, the nucleic acid or peptidenucleic acid is capable of hybridizing to nucleic acid or peptidenucleic acid analyte. In addition, the capture agent can be an aptamer,a nucleic acid capable of binding to non-nucleotide analyte (e.g.,proteins, small organic molecules, or inorganic molecules). As usedherein, an aptamer can be either an RNA or a DNA chain composed ofnaturally occurring or modified nucleotides.

Suitable capture agents also include members of binding pairs. Suitablebinding pairs include, for example, biotin and avidin or biotin andderivatives of avidin (e.g., streptavidin and NEUTRAVIDIN™).

Capture agents can be bound to the surface or to the bead as describedbelow or by using standard techniques for attaching polypeptides,nucleic acids, and the like to surfaces.

Analytes

As used herein, the term “analyte” refers to, for example, the molecularstructure that is recognized by a capture agent. For example, the termanalyte can refer to the epitope recognized by an antibody, or caninclude that part of a ligand that is bound by a receptor. The termanalyte also includes larger molecules that contain a molecularstructure that is recognized by a capture agent. The analyte can be partof a cell, for example a cell surface protein. The analyte can be ananalyte of interest, chosen by the user (e.g., preselected). The analytecan be selected based on the ability to bind a capture agent ofinterest, for example in small molecule library screening.

As described herein, the present invention can be used to measure one ormore analytes of a panel of analytes. The panel of analytes can includeone or more analytes that are detected using a competition format asdescribed herein. The panel of analytes can include one or more analytesdetected using the sandwich assay format as described herein. In oneembodiment, each analyte is detected using a separate cartridge asdescribed below In order to test a panel of one or more analytes, asingle sample can be divided into two or more aliquots. Each aliquot canbe tested for a different analyte, for example, using a differentcartridge for each analyte to be tested. In this manner, panels ofdifferent analytes may be tested without requiring that multiple samplesbe acquired and/or that different types of apparatus be employed to testthe detect the different analytes.

In one embodiment, the analyte of interest is a small molecule. Smallmolecules include organic or inorganic molecules having a molecularweigh on the order of about 1000 g/mol or less. Typically, smallmolecule analyte will contain a single or only a few binding sites.Because a small molecule has a few or only one binding site, the presentinvention uses competitive binding to detect and/or quantify smallmolecule analytes.

It is understood, however, that some small molecules can be detected ina sandwich assay format. For example, the detection of rapamycin can beachieved by direct sandwich immunoassay with one capture agent orprotein interaction partner immobilized on the sensor surface and theother capture agent or protein partner on the magnetic bead. Twoproteins responsible for binding rapamycin are the 12-kD FK506 bindingprotein (FKBP) and the 100-amino acid domain of the mammalian target ofrapamycin (mTOR) known as the FKBP-rapamycin binding domain (FRB). Ithas been shown that FKBP and FRB do not exhibit apparent affinity towardeach other in the absence of rapamycin.

The small molecule can include, for example, steroids, lipids,carbohydrates, peptides, and heterocyclic compounds (e.g. bases,including co-factors such as FAD and NADH). The analyte (e.g., smallmolecule) can be part of a library of small organic molecules whichcomprise aldehydes, ketones, oximes, hydrazones, semicarbazones,carbazides, primary amines, secondary amines, tertiary amines,N-substituted hydrazines, hydrazides, alcohols, ethers, thiols,thioethers, thioesters, disulfides, carboxylic acids, esters, amides,ureas, carbamates, carbonates, ketals, thioketals, acetals, thioacetals,aryl halides, aryl sulfonates, alkyl halides, alkyl sulfonates, aromaticcompounds, heterocyclic compounds, anilines, alkenes, alkynes, diols,amino alcohols, oxazolidines, oxazolines, thiazolidines, thiazolines,enamines, sulfonamides, epoxides, aziridines, isocyanates, sulfonylchlorides, diazo compounds and/or acid chlorides, preferably aldehydes,ketones, primary amines, secondary amines, alcohols, thioesters,disulfides, carboxylic acids, acetals, anilines, diols, amino alcoholsand/or epoxides, most preferably aldehydes, ketones, primary amines,secondary amines and/or disulfides and combinations thereof.

In a particular embodiment, the analyte is estradiol. The term estradiolincludes estradiol and all measurable estradiol metabolites. The termestradiol can include; therefore, estrone-3-glucuronide (E3G),estradiol-3-glucuronide, estradiol-17-glucuronide,estriol-3-glucuronide, estriol-16-glucuronide and estrone-3-sulphate.

In one embodiment, estradiol levels can be used to determine the statusof ovarian reserves in a patient and/or predict fecundity in a patient.The present invention can be measured as part or a panel of analytes todetermine ovarian reserve status or fertility status. The analyte panelcan include, for example, estradiol and FSH. Methods for predicting areknown in the art, for example, as described in Buyalos, et al., FertilSteril 68:272-277, 1997, the teachings of which are incorporated hereinby reference in their entirety.

The analyte of interest can also be a biologic analyte, such as apolypeptide, a nucleic acid, a carbohydrate, a nucleoprotein, aglycopeptide or a glycolipid. Useful analytes include, for example,enzymes, steroids, hormones, transcription factors, growth factors,immunoglobulins, steroid receptors, nuclear proteins, signaltransduction components, allosteric enzyme regulators, and the like.Analytes of interest can be obtained, for example, commercially,recombinantly, synthetically, or by purification from a natural source.In preferred embodiments, the analyte of interest is associated with aspecific human disease or condition. Suitable growth factors include,for cytokines such as erythropoietin/EPO, granulocyte colony stimulatingreceptor, granulocyte macrophage colony stimulating receptor,thrombopoietin (TPO), IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-11, IL-12,growth hormone, prolactin, human placental lactogen (LPL), CNTF, andoctostatin. Suitable steroids include, but are not limited to,estradiol, progesterone, testosterone, and derivatives thereof. Othersuitable analytes include, for example, insulin, insulin-like growthfactor 1 (IGF-1), epidermal growth factor (EGF), vascular endothelialgrowth factor (VEGF), placental growth factor (PLGF), TGF-α and TGF-β),other hormones and receptors such as bone morphogenic factors, folicalstimulating hormone (FSH), and leutinizing hormone (LH), tissue necrosisfactor (TNF), apoptosis factor-1 and -2 (AP-1 and AP-2), and mdm2. Abiologic analyte also includes cellular analytes that include, forexample, a suitable detectable marker, such as a surface receptor.

In one embodiment, the analyte is a breast cancer marker. Breast cancermarkers include for example, estradiol and metabolites thereof asdescribed above. In addition, breast cancer makers include, but are notlimited to proteins described in U.S. Pat. No. 6,936,424 to Watkins, etal., the teachings of which are incorporated herein in their entirety.

The analyte of interest can also be a therapeutic drug where it would beuseful to measure the levels of the drug in a patient sample, forexample for drug management purposes. Suitable therapeutic drugsinclude, but are not limited to protease inhibitors andimmunosupressants. Suitable protease inhibitors include ageneraser,reyataz, lexiva, telzir, crixivan, kaletra, viracep, norvi, invirase,aortovase, aptivus and the like). Suitable immunosupressants includecyclosporin, tacrolimus (FK-506), rapamycin, mycophenolic mofetil andthe like. In one embodiment, the therapeutic drug of interest isdetected using a competition assay.

In another embodiment the therapeutic drug is detected in a sandwichassay format. As described above, the therapeutic drug can be bound to acarrier or binding protein and can be detected in a sandwich assay usinga capture agent that is capable of binding the carrier. In anotherembodiment, the therapeutic drug is a biological therapeutic. Examplesof biological therapeutics include: monoclonal antibodies, enzymes,hormones and other proteins. Since these molecules are much larger, theygenerally have multiple epitopes and can be detected in a sandwichassay.

Transplant patients are usually prescribed a combination ofimmunosuppressant drugs. For example, calcineurin inhibitors, such ascyclosporine and tacrolimus, are often prescribed together withmycophenolic mofetil. Additionally, rapamycin (sirolimus) can be usedalone or in combination with tacrolimus and cyclosporine. As such, it isbeneficial to provide a panel of measurements from a single patient toaid the physician in determining modifications to their drug levels. Thepanel of analytes can include small molecules and biologicals (alsoreferred to herein as biomarkers). The analyte panel can include, forexample, one or more of the prescribed therapeutic drugs and optionallyan immune response marker, such as one or more inflammatory cytokines.Furthermore, it is possible to combine the measurements of therapeuticdrugs and biomarkers to monitor infection or rejection. Similarstrategies could be used for other patient conditions and therapeutics.

The analyte of interest can be a pathogen or microbe, such as bacteriaor bacterial spores, viruses, parasites, prions or other pathogens ortheir cell wall or surface components such as gram-positivepeptidoglycans, lipoteichoic and teichoicacids, and gram-negativeendotoxin (e.g.) lipopolysaccharide). Bacterial analytes include, forexample, Shigella sp. such as Shigella dysenteriae, Campylobacter sp.such as Campylobacter jejuni, Enterococcus sp. such as Enterococcusfaecalis, Bacillus anthracis, Yersinia pestis, Bordetella pertussis,Streptococcal species, Staphylococcus aureus, Mycobacteriumtuberculosis, Clostridium difficile, Clostridium tetani, Clostridiumbotulinum, Escherichia coli, Salmonella thyphimurim, Salmonellaenterica, Chlamydia species, Treponema pallidum, Neisseria gonorrhoeae,Borrelia burgdorferi, Vibrio cholerae, Corynebacterium diphtheriae, andHelicobacter pylori. Parasites include, for example, Giardia, malariaand crytosporidia. Viral analytes include, for example, Rhinovirus,Yellow Fever, Group B Coxsachieviruses, (CB1, CB2, CB3, CB4, CB5, andCB6), Canine Parvovirus (CPV), Herpes Simplex virus type 1 (HSV 1),Vaccina Virus, T4-like virus, Adenovirus, Influenza B virus, InfluenzaA, Avian flu, rhinovirus, coronavirus (e.g., SARS), HumanImmunodeficiency virus (HIV), Hepatitis viruses, Herpes virus, West NileVirus, and Ebola virus.

As described above, in some embodiments, more than one analyte isdetected. In one embodiment, the analytes have the same molecularstructure. The analytes can be, for example, the same molecular speciesof interest. In another embodiment, the different analytes (alsoreferred to herein as the first and second analytes) are part of alarger molecule. Therefore, the analyte can be a particular binding site(such as an epitope) attached to or contained within a larger molecule.As such, the different analytes being detected can be part of differentmolecules.

Analytes can be bound to the surface or to the bead as described belowor by using standard techniques for attaching small molecules,polypeptides, nucleic acids, and the like to surfaces. In oneembodiment, the analyte is indirectly bound to the surface. The analytecan be indirectly bound to the surface, for example, by coating thesurface with a first member of a binding pair. The analyte is bound orattached to a second member of the binding pair and then the analyte isbound to the surface via the interaction between the first and secondmembers of the binding pair. Suitable binding pairs include, forexample, biotin and avidin or biotin and derivatives of avidin such asstreptavidin and NEUTRAVIDIN™.

According to one embodiment of the method of the present invention, theplurality of particles can be exposed to the sample using a variety ofdifferent formats. For example, in one embodiment, the plurality ofparticles is exposed to the sample and then introduced into the fluidchamber. The sample may be concentrated prior to introducing thesample-exposed particles into the fluid chamber. The sample may beconcentrated, for example by removing the beads from the solution andresuspending in a smaller volume of liquid.

In another embodiment, the sample is introduced into the fluid chamberprior to introducing the plurality of particles. In still anotherembodiment, the plurality of particles is introduced into the fluidchamber prior to adding the sample to the fluid chamber.

In another embodiment, as described above, the plurality of particles isexposed to the sample and to a competitor molecule. The plurality ofparticles can exposed to the sample and to the competitor molecule in anumber of different formats. For example, in one embodiment, theplurality of particles is exposed to the sample and to the competitormolecule and then introduced into the fluid chamber. In anotherembodiment, the sample and the competitor molecule are introduced intothe fluid chamber prior to introducing the plurality of particles. Instill another embodiment, the plurality of particles is introduced intothe fluid chamber prior to adding the sample and/or the competitormolecule to the fluid chamber.

As described above, in one embodiment of the present invention, aplurality of magnetic particles and a competitor molecule are introducedinto a fluid chamber. The magnetic particles are coated with a firstcapture agent capable of binding the analyte and at least one surface ofthe fluid chamber comprises an acoustic device that has been coated witha second capture agent capable of binding to the competitor molecule. Inone embodiment, the competitor molecule comprises the analyte linked orbound to a tag, and the second capture agent is capable of binding tothe tag. The tag can be any moiety that can be recognized by a captureagent. In one embodiment, the tag is one member of a binding pair andthe capture agent is the other member of the binding pair. For example,the tag can be biotin, and the capture agent can be avidin,streptavidin, or NEUTRAVIDIN™. In this embodiment, the biotin is linkedto the analyte using known methods for linking molecules to biotin, toform the competitor molecule. The surface can be coated with the secondmember of the binding pair using any suitable method. For example, thesurface can be coated with biotin as described below and then thebiotinylated surface can be exposed to avidin or a derivative of avidin.

In another embodiment, the competitor molecule comprises two or moreanalyte molecules bound to a carrier, and the second capture agent iscapable of binding the analyte. The carrier can be any molecule to whichtwo or more analyte molecules can be linked or bound. The carrier can bea protein, a nucleic acid, or other polymer. In one embodiment, thecarrier is an albumin, such as bovine serum albumin. In anotherembodiment, the carrier is horseradish peroxidase. In this embodiment,two or more analyte molecules can be linked to the carrier as describedbelow, or known linking technology can be used to form the competitormolecule. The surface can be coated with the capture agent as describedbelow.

As described above, the present invention can be used to screen a smallmolecule library for one or more small molecules of interest. Smallmolecule libraries of the type useful in methods of the presentinvention may be formed by methods well known in the art or may beobtained commercially (e.g., from ChemBridge on the world wide web atchembridge.com). Small molecule libraries include combinatoriallibraries. A combinatorial library can be a library of moleculescontaining a large number, typically between 10³ and 10⁶, ofdifferent-sequence oligomers, typically characterized by differentsequences of subunits, or a combination of different sequences of sidechains and linkages, or different-substituent compounds. Varioussolid-phase or solution-phase synthetic methods for preparing smallmolecule libraries are known. In one approach, beads containingsuccessive precursors to the target compounds that form the library arealternately mixed and separated, with one of a selected number ofreagents being added to each group of separated beads at each step(Furka, et al., Int. J. Pept. Protein Res. 37:487-493 (1991); Chen etal., J. Am. Chem. Soc. 116:2661-2662 (1994); Pham, et al., WO 9513538(1995); Dillard, et al., WO 9408051 (1994). In this embodiment, eachbead contains only one chemical species. The plurality of particles canbe prepared by synthesizing analytes on a surface of each particle usingknown methods as described above. In one embodiment, a different analyteis synthesized on each particle.

In one embodiment, the method comprises preparing the plurality ofparticles having different analytes bound thereto. In one embodiment,the plurality of particles includes two or more groups of particles,wherein each group of particles is coated with a different analyte. Theparticles can be prepared by exposing separate groups of particles todifferent analytes such that the particles in the group become coatedwith the different analytes. The groups of particles can then be used inthe method of the present invention or can be mixed together prior touse in the method of the present invention.

Control Signal/Normalization

In another embodiment, the signal output of the acoustic device inresponse to sample exposure is compared to or normalized with a controlsignal. The control signal can be provided to or obtained by the user.For example, the control signal can be a value provided to the userbased on the specific analyte, capture agent, or particular model,version or type of device being used. In one embodiment, the controlsignal is obtained on a lot basis. For example, the control signal canbe a signal that is representative of a particular lot of analyteacquired by the user. The representative signal can be, for example,experimentally derived before, during or after testing of a sample. Insome embodiments, the control signal is a standard curve that isobtained by, for example, analyzing known quantities of analyte with aspecific capture agent and specific version of an acoustic device.

In another embodiment, the control signal is obtained on a use basis.For example, a unique control signal can be obtained each time aparticular analyte and/or capture agent is tested on a particularacoustic device. In one embodiment, the control signal is obtained usingthe same analyte and/or capture agent, however, in the absence ofsample. The control signal can be obtained by introducing a secondplurality of particles into the fluid chamber that are coated with acapture agent. At least one surface of the fluid chamber comprises anacoustic device that has a second analyte bound thereto. Signal outputby said acoustic device is monitored to determine a control signal to beused in subsequent testing.

Acoustic Devices

Acoustic devices couple to fluids predominantly through acousticinteraction between the device and the fluid. Typical acoustic devicesinclude surface acoustic wave devices, flexural plate wave devices, lambwave devices and cantilever devices. Acoustic devices also couple tofluids through some viscous interaction between the device and thefluid, however, the coupling is predominantly acoustic coupling. Viscousinteraction devices couple to fluids predominantly through viscousinteraction between the devices and the fluid. Typical viscousinteraction devices include quartz microbalance (QCM) devices, shearharmonic surface acoustic wave devices, and acoustic plate mode devices.The term “surface acoustic wave” refers to the manner in which energy iscarried in the device structure rather than how the device couples tothe fluid. Acoustic devices are devices where fluid interacts over asubstantial area of a plane of the device. Acoustic devices respond withsubstantial out of plane motion that couples acoustically to fluid inproximity to the plane of the device (i.e., kinetic energy, potentialenergy and losses are carried predominantly in the fluid). Viscousinteraction devices respond primarily with in-plane motion that does notcouple acoustically to fluid in proximity to a plane of the device.

General Considerations

For applications involving, for example, the detection andquantification of biological or chemical substances in a fluid, thecoupling between an acoustic device and a fluid is typically betweenabout 100 nm and about 10 microns in thickness relative to the plane ofthe device where the coupling between a viscous interaction device and afluid is between about 10 nm and about 100 nm in thickness relative tothe plane of the device.

Surface acoustic wave devices and shear harmonic surface acoustic wavedevices both carry energy in their respective structures in similarmanners. Surface acoustic wave devices acoustically couple significantlyto fluids while shear harmonic surface acoustic wave devices couple tofluids predominantly through viscous interaction.

One embodiment of an analyte detection system 100, constructed accordingto the invention, is shown in FIG. 1A. The system 100 includes a networkof channels 102 for transporting various test solutions (also referredto herein as “test fluids” or “fluids”) through an FPW device 104. Thefollowing U.S. patents and patent applications, all of which are herebyincorporated by reference, describe examples of the various types of FPWdevices suitable for use in the present invention: U.S. Pat. No.5,129,262, U.S. Pat. No. 5,189,914, U.S. Pat. No. 6,688,158 B2, U.S.patent application Ser. No. 10/324,685, U.S. Pat. No. 5,668,303, U.S.Pat. No. 5,836,203, and U.S. Patent Application 20040038195.

For example, U.S. Pat. No. 5,129,262 describes an ultrasonic sensor thathas a thin planar sheet of material forming a Lamb wave propagationmedium. Lamb waves, also known as plate-mode waves, can propagate onlythrough a material of finite thickness. In contrast to surface acousticwaves (SAWs), which require a propagation medium having a thickness onthe order of hundreds of times the wavelength of the propagating SAW,Lamb waves require a propagation medium which is at most only severalwavelengths thick, and typically only a fraction of the wavelength ofthe propagating Lamb wave. The thickness of the sheet is no greater thanabout twenty microns. A Lamb wave generator generates Lamb waves in theplanar sheet, and an output device produces an electrical signal thatrepresents the propagation characteristics of the Lamb waves propagatingalong the sheet. A measuring device measures selected characteristics ofthe output electrical signal. The planar sheet has some physicalcharacteristics that depend upon the value of a measurand acting on thesheet, and those physical characteristics consequently determine thepropagation characteristics of the Lamb waves that propagate along thesheet. Since the electrical signal from the output device represents thepropagation characteristics, the electrical signal also represents thevalue of the measurand acting on the sheet.

The Lamb wave device described in U.S. Pat. No. 5,129,262 can beemployed, for example, in biological sensing. The planar sheet describedabove can be pre-coated with antibody molecules, so that the frequencyof the device changes upon immersion in or contact with a liquid thatcontains the corresponding antigen. Antigen-antibody attachment at thesurface of the propagation medium acts to alter the wave velocity of theLamb waves in the sheet. The change in wave velocity causes theoscillation frequency to change in a delay line oscillator form of thedevice. Also, the sheet may be made of a porous and permeable material,allowing the coating of antibody molecules over a greater surface areaof the sheet and also allowing the antigen-containing liquid to beflowed through the membrane, in order to speed up the antigen-antibodyattachment. Other biological interactions may also be sensed, andadditional applications include immunoassay, clinical laboratorytesting, in vivo biomedical monitoring, and biomedical research.

The test solutions used in the described embodiment, for example ablocking solution 106, a sample 108, and a buffer 110, are sourced fromreservoir containers 112. The channel path from each of the reservoirs112 is gated with a valve 114 to control the flow of a particular testsolution to a combination point 116 leading to an entry port 118 of theFPW device 104. The test solution flows through the FPW device 104 andexits via an exit port 120, which leads to a pump 122. The pump 122draws the test solution through the network of channels 102 and throughthe FPW device 104, and directs the test solution to a waste receptacle124.

FIG. 1B shows another embodiment of the analyte detection system 100.This embodiment packages the FPW device 104 and its associated fluidchamber 160 as a cartridge 103, i.e., a consumable component that can beremoved and replaced. Some embodiments may include a fluid controldevice 101 such as a plug, obstruction or baffle that alters the flowthrough the device 104. In one embodiment, the fluid control device 101operates to cause the fluid flow through the device 104 to pass closerto the sensor surface 143 than if the fluid control device 101 was notpresent. Further, the source of input test solutions is shown as aninput fluid chamber 105 that has an outlet 107 for directing the testsolutions into the inlet 109 of the cartridge 103. In some embodiments,the magnetic particles are initially located in the input fluid chamber105 and the fluid containing analyte is mixed with the magneticparticles in the input fluid chamber 105 and then directed into thecartridge 103 in which the FPW device 104 is located. The magneticparticles may be combined within the input fluid chamber 105 with thefluid containing analyte by a device (e.g., by the action of a pump or amagnetic agitator). FIG. 1B further shows an output fluid chamber 111with an inlet 113 that receives fluid from the outlet 115 of thecartridge 103. This output fluid chamber 111 may include one or more ofthe fluid control devices described herein, and it may include one ormore mechanisms for storing and/or treating waste fluid.

In at least one embodiment, the junction where the outlet 107 of theinput fluid chamber 105 meets the inlet 109 of the cartridge 103 isconstructed and arranged to allow repeatable connection anddisconnection. Similarly, the junction where the outlet 115 of thecartridge 103 meets the inlet 113 of the output fluid chamber 111 isconstructed and arranged to allow repeatable connection anddisconnection. In some embodiments, these junctions are constructed andarranged to require tools for connection and disconnection, such asthreaded couplings that require a wrench or other such tool to affectthe coupling and decoupling. In other embodiments, these junctions areconstructed and arranged to allow quick and easy manual connection anddisconnection, without any extra tools or accessories. Such couplings,both requiring and not requiring tools, are known in the art. In someembodiments, there are multiple input fluid chambers and output fluidchambers. In some embodiments, one or more input and/or output fluidchambers are part of the cartridge 103. Further, in some embodiments,one or more sources of magnetic flux are part of the cartridge.

The FPW device 104 is shown in more detail in FIG. 2 and FIG. 2A. In anFPW device 104, strain energy is carried in bending and tension in thedevice. In some embodiments, it is desirable for thethickness-to-wavelength ratio of the FPW device 104 to be less than one,and in some cases much less than one. In general, the wavelength “λ” ofthe FPW device 104 is approximately equal to the pitch of theinterdigitated electrodes as described herein. In one embodiment, thethickness-to-wavelength ratio of the FPW device 104 is 2 μm/38 μm. Inother embodiments, the FPW device 104 is designed to isolate aparticular mode (e.g., any mode from the zero^(th) order mode to higherorder modes) or bandwidth of modes associated with the device. Forexample, an FPW device 104 having a thickness/wavelength of 2 μm/38 μmas described above would isolate the 80^(th) mode of the FPW device 104.The FPW device 104 can be designed to achieve this effect by selecting aparticular pattern for the interdigitated electrodes deposited on thedevice. In one embodiment, the FPW device 104 is rectangular in shape.The FPW device 104 can, alternatively, be circular or elliptical, orsome other planar shape.

In general, the FPW device 104 is constructed from a silicon wafer 130,using micro-fabrication techniques known in the art. In the describedembodiment, a cavity 132 is etched into the wafer 130 to produce a thin,suspended membrane 134 that is approximately 1.6 mm long, 0.3 mm wideand 2 μm thick. The overall wafer 130 thickness is approximately 500 μm,so the depth of the cavity 132 is just slightly less than the wafer 130thickness. A 0.5 μm layer 136 of aluminum nitride (AlN) is deposited onthe outer surface (i.e., the surface opposite the cavity 132) of themembrane 134, as shown in the expanded view insert of FIG. 2A. Two setsof inter-digitated metal electrodes 138 are deposited upon the AlNlayer. A thin layer 140 of gold (approximately 500 angstroms) isdeposited on the inner surface (i.e., the surface facing the cavity 132)of the membrane 134 to facilitate immobilization of capture agents(described in more detail below).

In operation, instrument/control electronics 126 (referring to FIG. 1A)apply a time-varying electrical signal to at least one set of electrodes138 to generate vibrations in the suspended membrane 134. Theinstrument/control electronics 126 also monitor the vibrationalcharacteristics of the membrane 134 by receiving a sensor signal from atleast a second set of electrodes 138. When liquid is in contact with thecavity side 132 of the membrane 134, the maximal response of the platestructure is around 15-25 MHz. The instrument/control electronics 126compare a reference signal to the sensor signal from the second set ofelectrodes to determine the changes in the relative magnitude and phaseangle of the sensor signal as a function of frequency. Theinstrument/control electronics 126 interpret these changes to detect thepresence of the targeted analyte. In some embodiments, theinstrument/control electronics also determines, for example, theconcentration of the targeted analyte on the inner surface of themembrane 134.

Capture agents targeting the analyte of interest are immobilized on thethin layer of gold 140 covering the inner surface of the membrane 134,as described above. The surface can be coated with a suitable linkingcompound. Suitable linking compounds are commercially available. In oneembodiment, the linking compound comprises biotin PEG disulfide, asdescribed below. In another embodiment, thiol-terminated alkyl chainsare linked to the gold surface forming a self-assembled monolayer (SAM).A fraction of the SAM chains are terminated with reactive groups (e.g.,carboxyl) to allow covalent linking of capture agents to the SAM chainsusing biochemical process steps known in the art. The remainder of theSAM chains are terminated with non-reactive groups, preferably ones thathave a hydrophilic character to resist nonspecific binding (e.g.,oligomers of ethylene glycol). Other surface chemistries are describedin the literature and can be used to produce a capture surface.

The FPW device 104 is packaged to allow electrical connections to theelectrodes 138 on the outer surface of the membrane 134. Additionally,the FPW device 104 is mechanically supported by a channel block 142, toallow for the inner surface of the membrane 134 to contact the testsolutions and an interface is provided for contacting the sensor surface143 with the liquid sample. The channel block 142 creates a path (fluidchamber 160) for the test solutions to flow from an input port 118, pastthe inner surface of the membrane 134 and then out of an exit port 120.A seal 144 is formed between the FPW device 104 and the channel block142 to prevent test solutions from escaping from the channels 102 formedwithin the combination of the FPW device 104 and the channel block 142.The channel block 142 thus forms a fluid chamber, of which the FPWdevice 104 comprises one of the interior walls.

The channels 102 through the combination of the FPW device 104 and thechannel block 142 are approximately 0.5 mm in diameter. The channelblock 142 can be formed from a variety of materials, including plastic,metal or ceramic, among other materials.

The system 100 includes one or more fluid control devices for changingat least one fluid property, such as flow, pressure, or trajectory toname a few, within the system 100. The pump 122 and valves 114 shown inFIG. 1A that direct and control the flows of various test solutionsthrough the device and over the sensor surface 143 (as required toexecute a test protocol) are all examples of fluid control devices. Ingeneral, a fluid control device changes the at least one fluid propertyin the vicinity of at least one surface within the fluid chamber 160 ofthe device 104. Generally, this is done to distribute the magneticparticles along at least a portion of the sensor surface 143. Asdescribed above, in some embodiments the fluid control device is a pump(e.g., a peristaltic pump, centrifugal pump, rotary pump,electro-osmotic pump). In some embodiments, the pump is located on theentrance side of the fluid chamber, and in other embodiments the pump islocated on the exit side of the fluid chamber. In some embodiments, thedevice is a flow diverter (e.g., a plug, obstruction wall or baffle)that is disposed relative to the fluid chamber to alter the fluid flowin the vicinity of the at least one interior surface of the fluidchamber.

Referring to FIG. 1A, a single pump 122 is positioned on the waste sideof the FPW device 104. Suction that the pump 122 generates draws buffer110 or analyte in the sample 108 from their respective reservoircontainers 112 on the supply side of the FPW device 104. Valves 114 arepositioned on the supply side of the device 104 to control which testsolution is directed over the sensor surface 143 at any time during thetest protocol. The pump 122 controls the flow rate of the test.

A device for regulating temperature (e.g., a thermoelectric cooler) maybe associated with the FPW device 104 and channel block 142. Thisreduces the impact of variable environmental conditions on the FPWdevice 104 output by maintaining the device 104 at a relativelyconstant, known temperature. In an alternative embodiment, a temperaturesensor is included within the system 100, for example as part of the FPWdevice 104. The sensor signal from the FPW device 104 is scaled, at aspecific instant in time (or during a period of time), based on theoutput of the temperature sensor, in order to produce a signal that isindependent of the effects of temperature variations. This scaling couldbe done based on a mathematical model, or an analytical model, or somehybrid combination of a mathematical and analytical model.

In some embodiments of the system 100, a filter is included in the pathof the test solution to selectively filter particles (e.g., magneticparticles and biological materials) of a particular size to prevent themfrom entering the fluid chamber. By way of example, a particular testingprotocol may include steps for changing the filter during the test. Thiswould allow different types (i.e., sizes) of analytes and magneticparticles to be directed into the fluid chamber, and thereby tested bythe system 100, during different portions of the test.

In one embodiment, magnetic particles (e.g., paramagnetic orsuper-paramagnetic beads or microspheres), which have their surfacescoated with a capture agent, are mixed with a sample containing theanalyte. After a prescribed mixing time analyte-particle complexes 146result as do particles 147 that have bound nonspecific materials andparticles 148 that have bound nothing. The particles 146, 147 and 148are located in the sample reservoir 112.

The system 100 further includes a magnetic field inducing structure 150for producing magnetic flux in the vicinity of the membrane 134. In FIG.1A, the source of magnetic flux is a retractable magnet 150 arranged tonormally be in close proximity to the membrane 134 of the FPW device104. When the magnet 150 is in close proximity to the membrane 134, themagnet 150 produces a significant gradient magnetic field in thevicinity of the membrane 134. Under control of the instrument/controlelectronics 126, the retractable magnet 150 can be retracted away fromthe membrane 134 by a distance sufficient to substantially reducemagnetic fields in the vicinity of the membrane 134. In one embodiment,when in close proximity to the membrane 134, the magnet 150 is situatedapproximately 200 μm from the sensor surface 143 of the membrane 134. Inanother embodiment, when in close proximity to the membrane, the magnet150 is situated between about 50 μm to about 100 μm from the sensorsurface 143 of the membrane 134.

When the magnet 150 is in close proximity to the membrane 134, themagnet 150 provides a source of magnetic flux to draw the magneticparticles from the sample to the sensor surface 143. Theanalyte-particle complexes 146, as well as particles 147 withnonspecifically bound material and particles 148 with nothing bound,migrate from the liquid sample until they encounter the sensor surface143. The analyte binds with the capture agent on the sensor surface 143.Thus, the analyte forms a link between the magnetic particle and sensorsurface. The particles 147 with non-specifically bound material andparticles 148 with nothing bound are held at the sensor surface 143 bythe magnetic field. Additionally, weak binding forces can act betweenthe particles 146, 147, and 148 and the sensor surface 143. During thewash step of the protocol (described in more detail below), the magnet150 is retracted to reduce the magnetic force experienced by theparticles that have accumulated at the sensor surface 143. The wash flowrate is increased to remove particles 147 and 148 that are not bound tothe surface by analyte. Since the particles 147 with nonspecificallybound material as well as particles 148 with nothing bound are moreweakly linked to the sensor surface 143 than the analyte-particlecomplexes 146, they release from the sensor surface 143 at a lower washflowrate (and corresponding hydrodynamic force). Hence, removing themagnet 150 (i.e., substantially reducing the magnetic force experiencedby the particles 146, 147, and 148 at the sensor surface 143) is used todistinguish between particles with analyte 146 from those without(particles 147 and 148). One technique for engaging and retracting themagnet 150 is to mount it on a carriage (not shown) that is actuated bya cam system (not shown).

The magnet 150 material, geometry and distance from the sensor surface143 determine the field shape and field gradient, and therefore, theforce that the analyte-particle complexes 146 experience. High strengthpermanent magnets for use as the retractable magnet 150 are availablecommercially. For example, 1 mm diameter cylindrical NdFeB magnets canbe purchased from several vendors (e.g., Dexter Magnetic Technologies).In one embodiment, a 1 mm diameter and 5 mm long NdFeB magnet 150 ispositioned within 0.1 mm of the sensor surface 143 when engaged. Whenretracted the magnet 150 is at least 0.5 mm from the sensor surface 143.Since the membrane 134 of the FPW device 104 is very thin (2 μm) andmade of nonmagnetic materials (e.g., silicon, aluminum nitride or gold),the membrane 134 does not significantly perturb the magnetic field onthe sensor surface 143 side of the device 104. As a result, very highmagnitude magnetic fields and large field gradients can be achieved, asis necessary for high collection efficiencies.

The sample flow rate through the channels 102 is determined (e.g.,specified by an operator) by the residence time necessary for goodcollection efficiency. The sample flow rate is adjusted so that theaverage velocity over the sensor surface 143 is between about 1 andabout 5 mm/s. With an iron oxide paramagnetic particle with a diameterof approximately 3 μm, collection efficiencies approaching 50% can beachieved.

Other configurations of the source 150 of magnetic flux (i.e., themagnet) may be used. For example, an electromagnet can be used insteadof a permanent magnet. The electromagnet includes pole pieces thatextend to focus the field flux near the sensor surface 143 of the device104.

Alternatively, a magnetizable material can be fashioned and positionedadjacent to the sensor surface 143 (within 0.1 mm), and a separatemagnet combined with an open face of the magnetizable material to inducea magnetic field in the magnetizable material. The magnetic fieldinduced in the material serves to locate desirable field gradients nearthe sensor surfaces 143. In this way, large, low cost magnets can beused, and a single magnet can be used to address multiple sensors,depending on the fashioning of the material. Examples of usefulmaterials for this purpose are pure iron, high mu metals such as alloy49 (high nickel content iron), sna silicon steels (1-2% silicontypical). An advantage of using such a magnetizable material with anassociated magnet is that the sensor assembly can be simplified,allowing lower cost manufacturing. A low precision actuator can be usedfor engaging and retracting the magnet since the magnet need onlycontact the ferromagnetic core or be fully withdrawn. In the describedembodiment where the magnet 150 is positioned in close proximity to thesensor surface 143, a higher level of precision is required to achievegood assay repeatability. Although there is some loss of field strengthwith this approach, it is still possible to design the overall system toachieve good capture efficiencies (e.g., >10%).

The shape of the tip of the field inducing structure (e.g., magnet orferromagnetic material) may be tailored to enhance and/or concentratethe field gradient at the surface. Since the size of the FPW device 104(e.g., 0.3 mm×1.6 mm) is typically smaller than conventionally formedmagnets or machined inductors, the portion of the field inducingstructure adjacent to the membrane 134 can be tapered to concentrate themagnetic field in one or more locations on the sensor surface 143.Tapering the tip acts to increase both the local field magnitude and thelocal field gradients. For example, a wedge-shaped tip is well suited tothe current FPW device geometry.

One embodiment of the system 100 includes an optional second source 150a of magnetic flux that opposes or partially opposes the first source150 of magnetic flux. This second source 150 a of magnetic flux can beused to dislodge some of the magnetic particles that have adhered to thesensor surface 143. It may, for example, dislodge magnetic particles 148that do not have any bound analyte; they would not be as stronglyattached to the sensor surface 143 as the particles 146 that do havebound analyte. In some embodiments, the first source 150 of magneticflux is turned off or moved away from the sensor surface 143 and then,the second source 150 a of magnetic flux is positioned relative to theat least one surface of the fluid chamber to selectively remove magneticparticles. This may be done, for example, to remove magnetic particles148 that do not have any bound analyte and therefore they are not asstrongly bound to the sensor surface 143. This would achieve a similareffect as increasing the flow of fluid to remove magnetic particles 148that do not have any bound analyte.

Controlling the distribution of the analyte-particle complexes 146 onthe surface 143 of the device 104 can improve the device performance,since the device 104 has a suspended membrane 134 and not all parts ofthe membrane 134 contribute equally to the moving mass of the detectableresonance. For example, the system 100 can be constructed and arrangedto distribute the analyte-particle complexes 146 within one third of theFPW device 104 width along the middle two-thirds of the centerline ofthe long axis of the membrane 134. Taking into account flow fieldeffects, the shape of the tip of the field-inducing structure (e.g.,magnet 150) can be such that the field magnitude and field gradientincrease in the direction of the flow over the sensor membrane 134. Thatis, analyte-particle complexes 146 in the downstream regions, where theboundary layer is partially depleted of analyte, experience a higherfield and field gradient than do analyte-particle complexes 146 in theupstream regions.

In general, the system 100 can be constructed and arranged toconcentrate magnetic particles in one or more particular regions of thesensor surface 143. The response of the device 104 may not be uniformover the sensor surface 143 due to characteristics of the fabricationmaterials or the specifics of the sensor design. Thus, high sensitivityregions of the device 104 may be non-uniform and asymmetrical withrespect to the long and short axis centerlines of the device 104. Thus,the tip of the field inducing structure may be shaped to concentratemagnetic particles in the region or regions of highest sensitivity.

Varying the flow rate through the device 104 can also be used to achievea more uniform coverage of analyte-particle complexes 146 for a givenmagnetic field distribution. For a given field, magnetic particlesinteract with the sensor surface 143 as determined by the bulk fluidflow rate, much like a ballistic object might fall in the presence ofthe gravity body force. In this case, however, the magnetic inducedforce dominates. By varying the flow rate, the analyte-particlecomplexes 146 can be caused to interact with the sensor surface 143 atsubstantially different locations along the stream-wise flow direction.Furthermore, as the magnetic particles pile up (a non-desirableoccurrence if they are to be exposed to the sensor surface 143) the flowcan be reversed and subsequently pulsed forward in order to pull thepile over and thus communicate more particles with the sensor surface143. In one embodiment of the system 100, selective location of themagnetic particles along the sensor surface 143 is achieved byselectively altering, over the course of the detection protocol, eitherone or both of the magnetic flux source and the property or propertiesof the fluid flow along the sensor surface 143.

One embodiment of the system 100 includes a device (e.g., optical,magnetic) for characterizing at least one property of the magneticparticles that are attached or attracted to the sensor surface 143. Thisdevice could be an integral part of the FPW device 104, or it could be apart of the magnet 150, or it could be a discrete component apart fromother components of the system 100. Such a device may be used to detectthe presence of the particles, and also to determine parameters relatedto the particle, for example, the size, quantity, concentration, ordensity of the particles that are attracted to the sensor surface 143.

One embodiment of the system 100 includes an identification device forallowing an operator or computer to identify the system 100 or aparticular component of the system for tracking usage of the system orcomponent. The identification device may include a symbol or image suchas a bar code, an identification number, or other identifying mark. Theidentification device may include an actual component, passive oractive, such as an RFID tag, an integrated circuit or other suchcomponent known in the art for providing identifying information. Manysuch devices are known in the art, although any contemplatedidentification device may be used.

A general detection protocol 200 for using an FPW device 104 incombination with analyte-particle complexes 146 for detection ofbiological analytes is shown in FIG. 3.

The first step 202 of the detection protocol 200 is acquiring andpreparing 202 the analyte sample. Various preparation processes may needto be performed prior to testing, depending upon the particular type ofanalyte being tested. For example, to detect microbes in food (e.g., E.coli in ground beef), a sample would be first mixed with enrichmentbroth, stomached, incubated and filtered. For detecting proteins inblood, the sample would first be filtered or centrifuged and the serumseparated. Specific examples of test protocols that include samplepreparation steps are described herein.

The next step 204 of the detection process is mixing affinity-coatedparamagnetic particles (i.e., beads) with the prepared analyte sample.Paramagnetic or super-paramagnetic particles are available commerciallyfrom a number of vendors (e.g., Dynal Biotech, Oslo, Norway). Typicaldiameters range from 50 nm to 10 μm, and such particles can be procuredalready coated with affinity agents (e.g., antibodies, proteins, andnucleic acid probes) targeting a variety of analytes (e.g., cells,proteins and nucleic acids). Alternatively, the particles can bepurchased with various reactive chemical groups (e.g., epoxy, carboxyl,and amine) in order to attach a capture agent of choice. Standardbiochemical protocols are available for this purpose.

The sample with paramagnetic particles added is agitated 206 for anamount of time determined by the particular analyte and capture agent.During this process, the particles bind with analyte so that the analyteis captured on the particles. In some cases, the sample can be testeddirectly at this point. But, in other cases it is advantageous toperform separation steps 208 to isolate the analyte bound to theparticles from the rest of the original sample. These separation steps208 reduce interference from other biological material in the assay.Manual or automated equipment for performing such separation steps isavailable commercially (e.g., Dexter Magnetic Technologies, DynalBiotech). The basic process uses a magnet to localize the paramagneticparticles on a surface so that the majority of the sample liquid can beaspirated. The magnet (e.g., magnet 150 of FIG. 1A) is then removed, andclean buffer solution is added to re-suspend the particles.

In one embodiment, a baseline step 210 is executed prior to testing theprocessed analyte sample with the FPW device 104. During the baselinestep 210, a reference solution 106 is flowed through the system torinse/block the sensor 104, and the instrument/control electronics 126excite the device 104 and records the resulting initial baseline signalfrom the device 104.

A sample transport step 212 follows the baseline step 210. The sample108 containing the analyte-particle complexes 146 is flowed over thesensor surface 143 with the magnet 150 engaged. Analyte-particlecomplexes 146 are collected on the sensor surface 143. After aprescribed volume of sample 108 has flowed through the device 104, themagnet 150 is retracted to release the particles 147 and 148 from thesensor surface 143 that do not have bound analyte, and the flow isswitched to a wash solution (e.g., buffer solution 110). The flow rateof the wash solution is increased to help remove loosely bound particles147 and 148, as well as other material in the sample that may have boundto the sensor surface 143.

An acquisition step 214 follows the sample transport step 212. Referencesolution 106 is again run through the device 104, and theinstrument/control electronics 126 excite the device 104 to acquire andrecord a final baseline signal from the device 104.

The system 100 determines the amount of analyte accumulated on thesensor surface 143 during the transport step 212 by comparing 216 theinitial baseline signal and the final baseline signal, which correspondto the vibrational characteristics of the FPW device 104 in thereference solution before and after, respectively, the acquisition step214. Analyte-particle complexes 146 bound to the sensor surface 143change the vibrational characteristics of the FPW device 104, and theamount of change to the vibrational characteristics correspond to theamount of analyte-particle complexes bound to the sensor surface 143.

FIG. 4 shows the change in the signal from multiple FPW devices 104 as afunction of time for an exemplary detection protocol. The square symbolscorrespond to a device 104 exposed to an analyte; the triangles andstars correspond to negative controls (in which there is no analyte onthe beads). The data shown in FIG. 4 (and FIG. 5 as described below)represent an exemplary detection protocol for which the analyte is E.coli bacteria, or generally a cellular analyte.

In this particular experiment, the frequency of a resonant peak wastracked. FIG. 4 shows that the resonant frequency of the FPW device 104decreases as the magnetic particles 146, 147 and 148 accumulate on thesurface. Once the magnet 150 is removed and some of the magneticparticles are washed away, the frequency of the device 104 increases.Eventually, the system establishes a final baseline that can be comparedto the initial baseline taken at the start of the test, or to that of acontrol.

FIG. 5 is a summary plot showing the final signal change detected as afunction of original analyte concentration.

Other detection protocols may be used with the system 100 describedabove. Individual steps can be eliminated or added depending on therequirements of a specific application or analyte. For example, FIG. 6shows a time evolution plot for a detection protocol, similar to the oneshown in FIG. 4. FIG. 6, however, depicts a detection protocol for a PSAassay, which demonstrates the capability of the system 100 for detectingproteins. Also, the flow direction can be reversed during the protocol.For example, reversing the flow direction may be useful for washingnonspecifically bound material from the device, or for making moreefficient use of the available sample.

Another variation in the detection protocol includes alternating betweenwash steps and binding steps. This can allow better use of the dynamicrange of the device. In come cases, a large fraction of the particles donot have bound analyte, especially at low analyte concentrations. Byrepeatedly binding and washing away particles, it is possible toaccumulate more analyte-particle complexes 146 and, hence, improve thesensitivity of the measurement.

Changing or manipulating the magnetic field distribution at the sensorsurface 143 during the transport step 212 can enhance the probabilitythat the analyte attached to a particular particle encounters the sensorsurface 143. For example, if the spatial distribution of the field isalternated during binding, it is possible to cause the paramagneticparticles at the sensor surface 143 to roll. In some embodiments, bycontrolling the spatial distribution of the field, an operator or theinstrument/control electronics 126 can be used to control the rolling ofthe paramagnetic particles along the sensor surface 143.

As described above, introducing a second magnetic field (i.e., a secondsource of magnetic flux) in the system 100 can improve the control ofthe assay conditions and enhance the specificity of the assay. Forexample, during the binding or wash steps of the protocol as describedabove, applying a secondary magnetic field to the sensor surface 143 canact to pull off weakly bound magnetic particles. The strength of thesecondary field can be tailored to generate a force on theanalyte-particle complexes 146 at the sensor surface 143 that is belowthe binding force of the specifically bound analyte but above thetypical binding force for nonspecifically bound material. This sequenceof steps can be repeated multiple times during the assay to furtherincrease the specificity of the test.

The relative binding strength of the various analyte-particle complexes146 on the sensor surface 143 can be determined by increasing(continuously or discretely) this magnetic pull-off force during thewash step, while monitoring the response of the FPW device 104.Information on the relative binding strength can be used to distinguishbetween different analytes in the sample 108.

The particular way the sample interfaces with the device 104 can bedifferent in other embodiments. In the above-described embodiments, thesystem 100 flows the sample through a channel to establish contactbetween the analyte-particle complexes 146 and the sensor surface 143.In an alternative variation of the system 100, the FPW device 104 ismounted on a probe and at least partially immersed into a test solutioncontaining magnetic particles bound to an analyte. For this embodiment,the immersion is sufficient to place the bound magnetic particles inproximity to the sensor surface 143 so that the particles are attractedtoward the sensor surface 143 and subsequently detected, as describedherein. To obtain a baseline signal, the device 104 (or cartridge 103)is immersed in a reference test solution. In some embodiments, a portionof the device 104 (e.g., the membrane 134) is mounted to a probe.Further, only part of the sensor surface 143 of the membrane 134 isplaced in contact with the solution containing magnetic particles boundto an analyte. In these embodiments, immersion or controlled movement ofthe probe in the fluid is sufficient to place the bound magneticparticles in proximity to the sensor surface 143 so that the particlesare attracted toward the sensor surface 143 and subsequently detected.

Another alternative embodiment of the system 100 involves mounting thedevice 104 inside of a tube that can be partially immersed into a wellholding the sample 108, and then retracted. A pump applies suction todraw sample into the tube and over the sensor surface 143 (or cartridge103) when the sample is immersed. The sample is then ejected back intothe well by reversing the pump or simply by venting the tube. This cycleof drawing and releasing the sample can be repeated to improve thecollection efficiency and, therefore, the performance of the assay.

The following examples illustrate, for one embodiment of the system 100described herein, steps for preparing and utilizing the system 100 fordetecting an analyte.

EXAMPLES Example I Generalized Method for Capture AgentFunctionalization of a Surface of a Flexural Plate Wave Device

1. Deposit gold onto the surface (e.g., sensor surface 143) of theflexural plate wave device 104 and clean the gold surface 143 with, forexample, oxygen plasma.

2. An ideal surface chemistry for the surface 143 of the gold is onethat provides 1) non-specific binding resistance and 2) reactive groupslocated on the surface for covalent attachment of capture agents. Anexemplary surface chemistry for the surface 143 of the gold is aself-assembled monolayer (SAM) of alkane thiols. The SAM can be formedfrom a mixture of two alkane thiols; one terminated with a reactivegroup for subsequent covalent attachment of capture agents, and oneterminated with a non-reactive group. By way of example, a mixture ofEG₃—OH (EG3) and EG₆—OCH₂COOH (EG6) terminated C₁₁-alkane thiols may beused for this purpose. In one embodiment, the flexural plate wave device104 (particularly the surface 143 of the device 104) is placed incontact with the alkane thiol solution and allowed to incubate at roomtemperature for, for example, about 16 hours. The surface 143 of theflexural plate wave device 104 is then rinsed with ethanol and blown drywith nitrogen.

3. The next step involves covalent attachment of capture agent oranalyte to the surface 143 of the flexural plate wave device 104. Anumber of methods may be used for covalent attachment of capture agentor analyte. An exemplary method involves covalently linking a biotinlinker moiety to the SAM, then binding a biotinylated antibody oranalyte to the flexural plate wave device surface 143 via a streptavidinlinking layer.

Example II Detecting E. coli 0157:H7 In Ground Beef Using, for Example,the Method of FIG. 3 (FIG. 5 Contains Data Representative of VariousConcentrations of E. coli)

1. Prepare an analyte sample containing E. coli O157:H7 with aconcentration greater than about 100 cfu/mL.

2. Concentrate the analyte sample in solution by performing animmunomagnetic separation. A variety of commercial instruments (e.g.,PATHATRIX™ (antibody coated paramagnetic particles) by MatrixMicrosciences and BEAD RETRIEVER™ (magnetic bead processor) by DynalBiotech) or manual methods may be used to perform the immunomagneticseparation. An exemplary manual method involves:

a. Resuspend magnetic beads coated with E. coli antibody (e.g.,DYNABEADS™ anti-E. coli 0157, available from Dynal Biotech) until themagnetic bead pellet in the bottom of the tube disappears. Place amicrocentrifuge tube in the rack (e.g., a Dynal MPC-S) of a magneticplate. Pipette 1-20 μL of magnetic bead stock solution into the tube(the volume of magnetic bead stock selected is based on desired finalbead concentration).

b. Add 1 mL of the analyte sample to the tube in the rack of themagnetic plate and close the tube.

c. Invert the rack a few times. Incubate the solution in the tube atroom temperature for 10 to 60 minutes with gentle continuous agitationto prevent magnetic beads from settling.

d. Invert the rack several times to concentrate the magnetic beads intoa pellet on the side of the tube. Allow about 3 minutes for properrecovery.

e. Open the tube and carefully aspirate and discard the samplesupernatant as well as the remaining liquid in the tube's cap.

f. Remove the magnetic plate.

g. Add 1 mL of wash buffer (PBS TWEEN® 20 (polyoxyethylene glycolsorbitan monolaurate)) to the tube. Close the cap of the tube and invertthe rack a few times to resuspend the beads.

h. Repeat steps d-g twice.

i. Mix the contents of the tube briefly using a vortex mixer.

3. Detection of E. coli O157:H7

a. Functionalize (similarly as described by the steps in A.3 and A.4)the surface 143 of a flexural plate wave device 104 with E. coli O157:H7antibody.

b. Place a first inlet hose into a tube containing standard wash buffer(1×PBS with 0.05% TWEEN® 20 (polyoxyethylene glycol sorbitanmonolaurate)) and a second inlet hose into the tube containing themagnetic beads bound with E. coli O157:H7 (prepared in B.2). The firstinlet hose and second inlet hose are joined by a t-joint so the twohoses are in fluid communication and fluid from either the first inlethose or from the second inlet hose is directed to an inlet of the fluidchamber 160. Each of the two hoses has a valve that is capable ofpermitting or limiting the flow of fluid through respective hoses.

c. Place a first outlet hose in fluid communication with an outlet ofthe fluid chamber 160. Fluid from the first outlet hose is collected ina waste collection bottle.

d. A baseline output signal is obtained using the flexural plate wavedevice. The baseline signal is measured with standard wash bufferflowing into the fluid chamber 160 and out of the fluid chamber 160 forabout 5 minutes at a standard pump speed (e.g., 200 μL/min).

e. The first source of magnetic flux 150 is engaged, and then fluid fromthe tube containing the analyte is directed into the inlet of the fluidchamber 160. Fluid is directed into the inlet of the fluid chamber 160to accumulate magnetic beads bound with E. coli O157:H7 on the at leastone surface 143 of the flexural plate wave device 160 until a desiredamount is attached to the at least one surface 143. In one embodiment,the desired amount is achieved when, for example, there is a frequencyshift in the output of the flexural plate wave device 104 of about 4000ppm.

f. The flow of fluid from the tube containing the analyte is thendiscontinued. Fluid from the wash buffer tube is then directed into thefluid chamber 160 to wash away nonspecifically bound material (i.e.,materials other than 1) magnetic beads and 2) magnetic beads with boundanalyte).

g. The first source of magnetic flux 150 is then disengaged.

h. Initiate automatic wash protocol to remove any magnetic beads ormatrix components.

i. The final signal output by the flexural wave plate device 104 is thenmeasured. The baseline signal is compared with the final signal todetermine the concentration of E. coli O157:H7 in the analyte sample.

Example III Detecting Prostate Specific Antigen (PSA) in Human BloodSerum Using, for Example, the Method Steps of FIG. 3.

1. Prepare an analyte sample containing human serum obtained bycentrifugation from a human blood sample.

2. Concentrate the analyte sample in solution by performing animmunomagnetic separation. A variety of commercial instruments (e.g.,PATHATRIX™ (antibody coated paramagnetic particles) by MatrixMicrosciences and BEAD RETRTEVER™ (magnetic bead processor) by DynalBiotech) or manual methods may be used to perform the immunomagneticseparation. An exemplary manual method involves:

a. Resuspend magnetic beads coated with PSA antibody (e.g., DYNABEADS™anti-PSA, available from Dynal Biotech) until the magnetic bead pelletin the bottom of the tube disappears. Place a microcentrifuge tube inthe rack (e.g., a Dynal MPC-S) of a magnetic plate. Pipette 1-20 μL ofmagnetic bead stock solution into the tube (the volume of magnetic beadstock selected is based on desired final bead concentration).

b. Add 1 mL of the analyte sample to the tube in the rack of themagnetic plate and close the tube.

c. Invert the rack a few times. Incubate the solution in the tube atroom temperature for 10 to 60 minutes with gentle continuous agitationto prevent magnetic beads from settling.

d. Invert the rack several times to concentrate the magnetic beads intoa pellet on the side of the tube. Allow about 3 minutes for properrecovery.

e. Open the tube and carefully aspirate and discard the samplesupernatant as well as the remaining liquid in the tube's cap.

f. Remove the magnetic plate.

g. Add 1 mL of wash buffer (PBS-TWEEN® 20 (polyoxyethylene glycolsorbitan monolaurate)) to the tube. Close the cap of the tube and invertthe rack a few times to resuspend the beads.

h. Repeat steps d-g twice.

i. Mix the contents of the tube briefly using a vortex mixer.

3. Detection of PSA

a. Functionalize (similarly as described by the steps in A.3 and A.4)the surface 143 of a flexural plate wave device 104 with PSA antibody.

b. Place a first inlet hose into a tube containing standard wash buffer(1×PBS with 0.05% TWEEN® 20 (polyoxyethylene glycol sorbitanmonolaurate)) and a second inlet hose into the tube containing themagnetic beads bound with PSA (prepared in B.2). The first inlet hoseand second inlet hose are joined by at joint so the two hoses are influid communication and fluid from either the first inlet hose or fromthe second inlet hose is directed to an inlet of the fluid chamber 160.Each of the two hoses has a valve that is capable of permitting orlimiting the flow of fluid through respective hoses.

c. Place a first outlet hose in fluid communication with an outlet ofthe fluid chamber 160. Fluid from the first outlet hose is collected ina waste collection bottle.

d. A baseline output signal is obtained using the flexural plate wavedevice 104. The baseline signal is measured with standard wash bufferflowing into the fluid chamber 160 and out of the fluid chamber 160 forabout 5 minutes at a standard pump speed (e.g., 200 μL/min).

e. The first source of magnetic flux 150 is engaged, and then fluid fromthe tube containing the analyte is directed into the inlet of the fluidchamber 160. Fluid is directed into the inlet of the fluid chamber 160to accumulate magnetic beads bound with PSA on the at least one surface143 of the flexural plate wave device 104 until a desired amount isattached to the at least one surface 143. In one embodiment, the desiredamount is achieved when, for example, there is a frequency shift in theoutput of the flexural plate wave device 104 of about 4000 ppm.

f. The flow of fluid from the tube containing the analyte is thendiscontinued. Fluid from the wash buffer tube is then directed into thefluid chamber 160 to wash away nonspecifically bound material (i.e.,materials other than 1) magnetic beads and 2) magnetic beads with boundanalyte).

g. The first source of magnetic flux 150 is then disengaged.

h. Initiate automatic wash protocol to remove any magnetic beads ormatrix components.

i. The final signal output by the flexural wave plate device 104 is thenmeasured. The baseline signal is compared with the final signal todetermine the concentration of PSA in the analyte sample.

Example IV PSA in Calibrator I

By way of illustration, an experiment was conducted in which data wasacquired using the system 100 of FIG. 1A, according to principles of thepresent invention. Dynal tosyl-activated super paramagnetic beads,functionalized with anti-Prostate Specific Antigen (PSA) captureantibody (PN 90205, Scripps Laboratories Inc. with offices in San Diego,Calif.) were exposed to samples. The samples comprised 1× PBS (PhosphateBuffered Saline) and 1% Bovine Serum Albumin (BSA), spiked withapproximately 0 pg/mL, 10 pg/mL, 100 pg/mL and 500 pg/mL of free PSA[Fitzgerald Industries International, Inc. with offices in Concord,Mass.]. A bead concentration on the order of approximately 2×10⁴beads/mL with respect to sample was used in the experiment. Spikedsamples where incubated with beads with gentle continuous agitation for1 hour.

Eight of the Flexural Plate Wave (FPW) devices 104 of FIG. 2 provided ona single chip in a cartridge (not shown) were functionalized withcomplimentary anti-PSA antibodies (PN 90197, Scripps Laboratories Inc.with offices in San Diego, Calif.) after being first primed with 1×PBScontaining 0.05% TWEEN® 20 (polyethylene glycol sorbitan monolaurate)[Sigma-Aldrich Co. with offices in St. Louis, Mo.].

Data was acquired and analyzed as described for example, in the U.S.Patent Application entitled “Methods and Apparatus for AssayMeasurements” by Masters et al. filed on May 2, 2006. A baselinemeasurement (similarly as described previously herein) of eightindividual frequencies, each corresponding to tracked sensor phases,each with respect to reference signals, was made at about 17800 seconds.Tracking phases are initially selected for each device to be within aresonance band of the device, near a frequency where the magnitude ofresponse is near a peak value and where the phase response hassignificant linear range with respect to frequency change. When trackingthe sensor phases, at each time point, individual device trackingfrequencies are found by 1) sweeping each device over a range offrequencies and recording the phase of response at each excitationfrequency with respect to a reference signal, 2) fitting a functionrelating excitation frequencies to measured phase for each device, and3) using that function to compute the tracking frequency correspondingto the previously determined tracking phase.

In this embodiment, the devices are operated near 20 MHz and the sweeprange is approximately 20 kHz. Over this range, the phase characteristicis substantially linear allowing the fit function to be linear. Thereference signal for each device comprises the output of a network ofpassive electrical components, resistors and capacitors, simultaneouslydriven by the excitation. The reference network is selected to match theattenuation and provide a preferred phase shift for the devices nearresonance. Baseline frequencies are referenced to, and normalized by,the tracked frequency and are shown as parts per million (ppm) at aselected point in time.

The sensing surface 143 of each device 104 was functionalized withcapture agent. Gold coated chips were cleaned using an oxygen plasmasource in which typical processing conditions were about 50 W for about2 minutes. The chips were subsequently immersed in pure ethanol for 30minutes. Next, the chips were transferred to a 0.5 mM solution of biotinPEG disulfide solution (Cat No. 41151-0895, POLYPURE™ AS with offices inOslo, Norway) in ethanol and allowed to incubate overnight. The chipswere transferred back into a pure ethanol solution for 30 minutes. Thechips received a brief, final ethanol rinse and were blown dry using anitrogen stream. Variations on preparation conditions can be made withsimilar results achieved.

The resultant biotinylated surface of the devices 104 was coated withNEUTRAVIDIN™ (PN 31000, Pierce Biotechnology, Inc. with offices inRockford, Ill.) by flowing a 10 μg/ml solution of NEUTRAVIDIN™ over thebiotinylated surface for 1 hour. Antibody was biotinylated according tothe manufacturer's instructions (PN F-6347, Invitrogen Corporation withoffices in Carlsbad, Calif.) and then coupled to the NEUTRAVIDIN™atedsurface, by flowing 5 μg/ml solution of the biotinylated antibody(diluted into 1×PBS 0.1% BSA buffer), over NEUTRAVIDIN™ coated surfacefor 1 hour.

PSA sample was introduced and simultaneously a magnetic field wasgenerated near the sensor surfaces 143 from about 17900 to about 18200seconds. Samples of approximately 0 pg/mL, 10 pg/mL, 100 pg/mL and 500pg/mL of free PSA were each provided to two different devices 104 (totalof eight devices). The samples were flowed over the sensors atapproximately 100 μL/min for a total sample run/volume of 500 μL flowingover the sensors. In this manner, Dynal tosyl-activated superparamagnetic beads coated with free PSA were bound to a substantialsurface (surface 143) of the devices 104. Each bead was bound to thesurface 143 of the devices 104 by a plurality of bonds. Each pluralityof bonds giving rise to the discriminatory force with which each bindsto the surface of the device. The characteristic of association anddissociation of an ensemble of beads for each sample determines theconcentration of analyte in that sample.

At approximately 18200 seconds the sample was replaced with primingbuffer (1×PBS, 0.05% TWEEN® 20 (polyethylene glycol sorbitanmonolaurate)). As shown in FIG. 11, a change (see location 30) in eachof the signals was observed from 18200 seconds to 18400 seconds andrepresents the change in bulk fluid properties associated with switchingfrom the sample fluids back to the buffer fluid. At approximately 18400seconds the magnetic field was disengaged. The flow speed of the bufferfluid (1×PBS containing 0.05% TWEEN® 20 (polyoxyethylene glycol sorbitanmonolaurate)) was approximately 50 μL/minute between about 18200 secondsand 18400 seconds (corresponding to a flow speed of approximately 1.5mm/second at the sensor surface 143).

The flow speed was increased over the next 450 seconds (from about 18400seconds to about 18850) from approximately 50 μL/min to approximately300 μL/min in linear increments (the flow speed was increased by about17 μL/min every 30 seconds for 450 seconds then the flow speed wasreduced to approximately 50 μL/min). In this manner, a controlledexternal influence (i.e., the flow speed in this embodiment) was appliedto the beads by increasing the flow speed between about 18400 and about18850 seconds). The portion of curves between about 18400 and about18850 seconds represents a signal that is indicative of the change inthe amount of beads (and other non-specific material) bound to thesensor surfaces 143 over that time period.

FIG. 11 is a graphical illustration of a plot of the data acquiredversus time. The Y-Axis of the plot is the change in relative magnitudeof the signal output by the device 104 (in parts per million) at atracked sensor phase near the resonance of the device 104. The X-Axis ofthe plot is time in units of seconds. The plot is the change in trackedfrequency referenced to and normalized by a tracked frequency at aselected point in time.

The data for each curve shown in parts per million of tracked frequency,was further normalized by the value of that curve at about 18350 secondsand referenced to the baseline frequency that was measured beforeintroduction of the sample fluid. Each data curve was thereby scaled toa value of 100% at about 18350 seconds compared to when sample isintroduced. The data associated with each of the normalized curves wasthen integrated with respect to time over the 450 seconds (from about18400 seconds to about 18850 seconds) after the magnetic field wasremoved at about 18400 seconds.

The integration was performed by accumulating respective device signallevels, each interval level multiplied by the respective interval timeperiods, and dividing the final sum by the period over which the sum wasperformed. This provides the time normalized amount of material elements(beads) bound to the surfaces 143 of the devices, and these values areshown here to be a measure of concentration of analyte associated witheach sample. In this manner, the concentration of analyte is determinedbased on the change in the amount of material elements bound to thesurfaces 143 of each device 104 during the period of time between about18400 seconds to about 18850 seconds. As shown in FIG. 11, approximately10 pg/mL of free PSA was detected within less than about 9 minutes ofintroducing the sample into the system.

In some embodiments, data points are omitted that are, for example,outside the normal variation observed in the curves. For example,spurious data points that vary by more than an order of magnitude invalue relative to adjacent data points may be omitted from subsequentanalysis. The data points may be removed from the data by, for examplean operator or by a computer program.

Example V Competitive Assay for Estradiol in Serum

A sheep monoclonal antibody specific for free estradiol was coupled toDynal M280-tosyl activated beads using standard methods.

A sensor surface was constructed by coupling disulphide-PEG-biotin tothe gold sensor surface, then coupling NEUTRAVIDIN™ (Pierce PN 31000)and then biotinylated antibody was performed as described above.

BSA coupled Estradiol, E2-6-CMO-BSA (Sigma PN E5630) was then coupled tothe antibody surface by flowing 10 ug/ml BSA coupled estradiol dilutedin 1× PBS buffer over the antibody surface for 1 hour. Since theestradiol is coupled to multiple amine sites on the BSA (30 mols ofestradiol per mol of BSA) the aforementioned approach provides for amoderately dense small molecule surface presented to solution.

Sample solutions comprising 70% charcoal striped human serum (TexasBiologicals), 30% 1×PBS 0.05% TWEEN® 20 (polyethylene glycol sorbitanmonolaurate), were made with free estradiol (Sigma) spiked in at 0pg/ml, 10 pg/ml, 30 pg/ml, 200 pg/ml. Beads, in concentration ˜24/mlwere incubated with sample for 2 hrs. The beads were then flowed overthe FPW using typical protocol and results obtained in approx 0.5 hrs.

In some samples danazol was also added as an decomplexing antogonist, toremove any interfering molecules such as Sex Hormone Binding Proteinfrom the estradiol. Approximately 10-100 ng/ml levels were added.

Sample was flowed over the FPW at approximately 70 μL/min with magneticfield engaged and beads were accumulated on the sensor surfaces. A washprotocol beginning at 50 μL/min, ending at 500 μL/min, with 50 μL/minintervals every 30 seconds was used to wash the bound beads off thesensor surfaces. FPW signals were normalized by exposure levels givingnormalized signals, and these signals were integrated over the washperiod. As shown in FIG. 12, less than 10 pg/mL can be detected in thepresence of the antagonist additive 36, and less than 50 pg/mL can bedetected in the absence of the antagonist additive 34.

As shown in FIG. 12, estradiol concentrations ranging from 1,000 pg/mLto 10 pg/mL were detected.

Example VI Competitive Assay for FK-506

1. FK-506 was added to 2 ml of buffer (0.05% 1×PBS TWEEN® 20(polyethylene glycol sorbitan monolaurate) containing 1% BSA) to produceanalyte samples with FK-506 concentrations of 0, 0:5, 2.0, and 20 ng/ml.

2. Eight μl of 1×10⁷/ml anti-FK-506 IgM coated beads prepared asdescribed above were added to each sample and incubated for 30-60 min.at room temperature. The beads are prepared by coupling anti-FK-506 IgM(Fitzgerald Industries Intl) to paramagnetic beads according to themanufacturer's instructions (Invitrogen Corporation). The analytesamples were concentrated in solution as described above.

3. Five hundred μl of a solution comprising carrier (HRP) labeled withFK-506 (Diasorin Inc.) were added to the sample and incubated for 60min. at room temperature.

4. The samples were analyzed using an FPW device where the sensor wasfunctionalized with capture agent (anti-FK-506 IgM). Referring to FIG.10, the sensor 12 was functionalized with NEUTRAVIDIN™ 30 as describedabove, and anti-FK-506 IgM was biotinylated 32 using standardbiotinylation methods. As shown in panel B of FIG. 10, analyte 10 (inthis instance FK-506) was mixed with competitor molecule 24 andparticles 16 labeled with capture agent 14 (in this instance anti-FK-506antibody). For this example, the competitor molecule 24 comprised thecarrier HRP labeled with the analyte FK-506). As shown in panels C andD, a lower level of FK-506 in the sample is expected to allow theparticles to bind to the competitor molecule and thereby to bind to thesensor surface. A higher level of FK-506 is expected to result in fewerparticles binding to the sensing surface because more of the particleswill be bound to FK-506 from the sample. As shown in FIGS. 13 and 14,FK-506 concentrations ranging from 0.5 ng/ml to 20 ng/ml were detectedin as little as 15 mm from introducing the sample into the device.

Example VII Competitive Assay for FK-506 in Processed Human Whole BloodMatrix

1. Four samples each of one 100 μl of blood was spiked with 0, 3, 10, or30 ng/ml FK-506. The drug was extracted from the blood matrix byperforming a protein digestion protocol according to the manufacturer'sinstructions (Diasorin Inc.).

2. Six hundred microliters of protein digestion reagent was added toeach sample.

3. The sample was mixed by vortexing, and incubated for 15 min. at roomtemperature (about 23° C.), producing a semi-transparent mixture.

4. The digestion reaction was stopped by incubating the samples at 75°C. for 35 min, producing a dark brown mixture.

5. The sample was mixed by vortexing and centrifuged at 1800 g for 10min, producing 500-600 μl of supernatant.

6. Five hundred microliters of supernatant was transferred into a newtube, the four samples the same concentration of FK-506 were combined inone tube, producing 2 ml of supernatant for each concentration ofFK-506.

7. The samples were analyzed as described above. As shown in FIGS. 15and 16, FK-506 concentrations ranging from 0.5 ng/ml to 20 ng/ml weredetected in as little as 8 min from introducing the sample into thedevice.

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The presentembodiments are therefore to be considered in respects as illustrativeand not restrictive, the scope of the invention being indicated theappended claims rather than by the foregoing description, and allchanges which come within the meaning and range of the equivalency ofthe claims are therefore intended to be embraced herein.

1. A method for detecting estradiol in a sample, the method comprisingthe steps of: a) introducing a fluid comprising a sample and a pluralityof magnetic particles coated with a first capture agent capable ofbinding estradiol into a fluid chamber, wherein at least one surface ofthe fluid chamber comprises a flexural plate wave device having a secondcapture agent capable of binding estradiol, wherein the flexural platewave device has a membrane that is capable of vibrating, and wherein thesecond capture agent is bound to the membrane; b) applying a retractablesource of magnetic flux positioned external to the fluid chamber closeto the membrane to create a significant magnetic field gradient at asurface of the membrane to attract magnetic particles in the fluidtoward the membrane; c) monitoring a first signal output by saidflexural plate wave device, wherein the first signal output is monitoredin the presence of a magnetic flux; d) after removing the retractablesource of magnetic flux, flowing a solution through the fluid chamber toremove magnetic particles not bound to the membrane; e) monitoring asecond signal output by said flexural plate wave device, wherein thesecond signal output is monitored in the absence of the magnetic flux;and f) comparing the first and the second signal output to a controlsignal, thereby detecting estradiol in the sample.
 2. The method ofclaim 1, wherein an increased level of estradiol is detected compared toa reference, which indicates that an individual from whom the sample isobtained has an increased likelihood of developing breast cancer.
 3. Themethod of claim 1, wherein prior to being introduced into the fluidchamber, said plurality of particles has been exposed to the sample. 4.The method of claim 1, wherein the sample has been introduced into thefluid chamber prior to introducing the plurality of particles.
 5. Themethod of claim 1, wherein a concentration of about 10 pg/ml isdetected.
 6. The method of claim 1, wherein the estradiol is selectedfrom the group consisting of free estradiol, sex hormone-bindingglobulin (SHBG)-bound estradiol, estrone-3-glucuronide, andestradiol-3-glucuronide.
 7. The method of claim 1, wherein the sample isselected from the group consisting of blood, serum, plasma, urine,saliva, and biopsy material.
 8. The method of claim 1, wherein thecontrol signal is a standard curve.
 9. The method of claim 1, whereinthe control signal is obtained without the sample.
 10. The method ofclaim 1, wherein said second capture agent capable of binding theestradiol is indirectly bound to said surface.
 11. The method of claim10, wherein said surface is coated with a first member of a bindingpair, and the second capture agent is bound to a second member of thebinding pair.
 12. The method of claim 11, wherein the first member ofthe binding pair is biotin.
 13. The method of claim 1, wherein the firstor second capture agent is an antibody.
 14. The method of claim 11,wherein the second member of the binding pair is a derivative of avidin.15. The method of claim 1, wherein a fluid flows across the membraneduring one or more of steps a), b), c) and e).